Methods and materials relating to stem cell growth factor-like polypeptides and polynucleotides

ABSTRACT

The invention provides novel polynucleotides and polypeptides encoded by such polynucleotides and mutants or variants thereof that correspond to a novel human secreted stem cell growth factor-like polypeptide. These polynucleotides comprise nucleic acid sequences isolated from cDNA libraries prepared from human fetal liver spleen, ovary, adult brain, lung tumor, spinal cord, cervix, ovary, endothelial cells, umbilical cord, lymphocyte, lung fibroblast, fetal brain, and testis. Other aspects of the invention include vectors containing processes for producing novel human secreted stem cell growth factor-like polypeptides, and antibodies specific for such polypeptides.

1. BACKGROUND

[0001] 1.1 Technical Field

[0002] The present invention provides novel polynucleotides and proteinsencoded by such polynucleotides, along with uses for thesepolynucleotides and proteins, for example in therapeutic, diagnostic andresearch methods. In particular, the invention relates to a novel stemcell growth factor-like polypeptide.

[0003] 1.2 Background Art

[0004] Identified polynucleotide and polypeptide sequences have numerousapplications in, for example, diagnostics, forensics, gene mapping,identification of mutations responsible for genetic disorders or othertraits, to assess biodiversity, and to produce many other types of dataand products dependent on DNA and amino acid sequences. Proteins areknown to have biological activity, for example, by virtue of theirsecreted nature in the case of leader sequence cloning, by virtue oftheir cell or tissue source in the case of PCR-based techniques, or byvirtue of structural similarity to other genes of known biologicalactivity. It is to these polypeptides and the polynucleotides encodingthem that the present invention is directed. In particular, thisinvention is directed to novel stem cell growth factor-like polypeptidesand polynucleotides.

[0005] Stem cells are defined as cells with the capacity for unlimitedor prolonged self-renewal that can produce at least one type of highlydifferentiated descendent. It is believed that between the stem cellsand its terminally differentiated progeny there is an intermediatepopulation of committed progenitors with limited capacity and restricteddifferentiation potential (Watt and Hogan, (2000) Science 287,1427-1430). Embryonic stem cell division and differentiation give riseto all the differentiated cells and organs of a multicellular organism.A reserve of stem cells is maintained during the adult life of anorganism in order to replenish the terminally differentiated cellpopulations like hematopoietic cells. It is generally assumed that theadult stem cells are derived from the embryonic stem cells and have onlya limited potential for differentiation. Stem cells in general have beenextremely difficult to culture and maintain in vitro, let alonedirecting them on a predetermined differentiation pathway.

[0006] However, more recently new research have shown that the adultstem cells do possess much wider potential for differentiation thanpreviously thought. It was shown that adult neural stem cells whentransplanted in an irradiated host, were able to populate the bonemarrow and give rise to myeloid, lymphoid and early hematopoietic cells(Bjornson et al, (1999) Science, 283, 534-537). Also, for the firsttime, researchers have been able to culture human embryonic stem cellsin vitro. The authors showed that human blastocyst cells can be culturedfor a prolonged time and could differentiate into variety of differentcell types (Thomson et al, (1998) Science, 282, 1145-1147). This hasopened the doors for using autologous transplantation and organregeneration for treatment of organ failures and degenerative diseases.Precise interactions of multiple receptors on the stem cells withsoluble and stromal cell expressed factors are required for a stem cellto divide and commit to differentiation. It has become apparent that thetissue niches and the microenvironment providing the factors are of theutmost importance. Cytokines like IL-3, IL-6, IL-7, and soluble proteinslike and flt-3, erythropoietin, and stem cell factor, all have beenshown to act in concert to achieve differentiation down a specificpathway. It is thought precise combinations of growth factors,cytokines, and tissue localization could give rise to differentdifferentiated stem cells populations.

[0007] Thus, the stem cell growth factor-like polypeptides andpolynucleotides of the invention may be used to induce differentiationof embryonic and adult stem cells to give rise to different cell types.They may also be used in the treatment of leukemia, hemophilia, anddegenerative diseases like Alzheimer's disease. The polynucleotides andpolypeptides of the invention may further be utilized to generate newtissues and organs that may aid patients in need of transplantedtissues.

2. SUMMARY OF THE INVENTION

[0008] This invention is based on the discovery of novel stem cellgrowth factor-like polypeptides, novel isolated polynucleotides encodingsuch polypeptides, including recombinant DNA molecules, cloned genes ordegenerate variants thereof, especially naturally occurring variantssuch as allelic variants, antisense polynucleotide molecules, andantibodies that specifically recognize one or more epitopes present onsuch polypeptides, as well as hybridomas producing such antibodies.Specifically, the polynucleotides of the present invention are based onpolynucleotides isolated from cDNA libraries prepared from human fetalliver spleen, ovary, adult brain, lung tumor, spinal cord, cervix,ovary, endothelial cells, umbilical cord, lymphocyte, lung fibroblast,fetal brain, and testis.

[0009] The compositions of the present invention additionally includevectors such as expression vectors containing the polynucleotides of theinvention, cells genetically engineered to contain such polynucleotides,and cells genetically engineered to express such polynucleotides.

[0010] The compositions of the invention provide isolatedpolynucleotides that include, but are not limited to, a polynucleotidecomprising the nucleotide sequence set forth in SEQ ID NO: 1-22, 24,26-27, 29, or 33; or a fragment of SEQ ID NO: 1-22, 24, 26-27, 29, or33; a polynucleotide comprising the full length protein coding sequenceof SEQ ID NO: 1-22, 24, 26-27, 29, or 33 (for example, SEQ ID NO: 23, 25or 28); and a polynucleotide comprising the nucleotide sequence of themature protein coding sequence of any of SEQ ID NO: 1-22, 24, 26-27, 29,or 33. The polynucleotides of the present invention also include, butare not limited to, a polynucleotide that hybridizes under stringenthybridization conditions to (a) the complement of any of the nucleotidesequences set forth in SEQ ID NO: 1-22, 24, 26-27, 29, or 33; (b) anucleotide sequence encoding any of SEQ ID NO: 23, 25, 28, 30-32, 34, or35; a polynucleotide which is an allelic variant of any polynucleotidesrecited above having at least 70% polynucleotide sequence identity tothe polynucleotides; a polynucleotide which encodes a species homolog(e.g. orthologs) of any of the peptides recited above; or apolynucleotide that encodes a polypeptide comprising a specific domainor truncation of the polypeptide comprising SEQ ID NO: 23, 25, 28, or31.

[0011] A collection as used in this application can be a collection ofonly one polynucleotide. The collection of sequence information orunique identifying information of each sequence can be provided on anucleic acid array. In one embodiment, segments of sequence informationare provided on a nucleic acid array to detect the polynucleotide thatcontains the segment. The array can be designed to detect full-match ormismatch to the polynucleotide that contains the segment. The collectioncan also be provided in a computer-readable format.

[0012] This invention further provides cloning or expression vectorscomprising at least a fragment of the polynucleotides set forth aboveand host cells or organisms transformed with these expression vectors.Useful vectors include plasmids, cosmids, lambda phage derivatives,phagemids, and the like, that are well known in the art. Accordingly,the invention also provides a vector including a polynucleotide of theinvention and a host cell containing the polynucleotide. In general, thevector contains an origin of replication functional in at least oneorganism, convenient restriction endonuclease sites, and a selectablemarker for the host cell. Vectors according to the invention includeexpression vectors, replication vectors, probe generation vectors, andsequencing vectors. A host cell according to the invention can be aprokaryotic or eukaryotic cell and can be a unicellular organism or partof a multicellular organism.

[0013] The compositions of the present invention include polypeptidescomprising, but not limited to, an isolated polypeptide selected fromthe group comprising the amino acid sequence of SEQ ID NO: 23, 25, 28,30-32, 34, or 35; or the corresponding full length or mature protein.Polypeptides of the invention also include polypeptides with biologicalactivity that are encoded by (a) any of the polynucleotides having anucleotide sequence set forth in SEQ ID NO: 1-22, 24, 26-27, 29, or 33;or (b) polynucleotides that hybridize to the complement of thepolynucleotides of (a) under stringent hybridization conditions.Biologically or immunologically active variants of any of the proteinsequences listed as SEQ ID NO: 23, 25, 28, 30-32, 34, or 35 andsubstantial equivalents thereof that retain biological or immunologicalactivity are also contemplated. The polypeptides of the invention may bewholly or partially chemically synthesized but are preferably producedby recombinant means using the genetically engineered cells (e.g. hostcells) of the invention.

[0014] The invention also provides compositions comprising a polypeptideof the invention. Pharmaceutical compositions of the invention maycomprise a polypeptide of the invention and an acceptable carrier, suchas a hydrophilic, e.g., pharmaceutically acceptable, carrier.

[0015] The invention also relates to methods for producing a polypeptideof the invention comprising culturing host cells comprising anexpression vector containing at least a fragment of a polynucleotideencoding the polypeptide of the invention in a suitable culture mediumunder conditions permitting expression of the desired polypeptide, andpurifying the protein or peptide from the culture or from the hostcells. Preferred embodiments include those in which the protein producedby such a process is a mature form of the protein.

[0016] Polynucleotides according to the invention have numerousapplications in a variety of techniques known to those skilled in theart of molecular biology. These techniques include use as hybridizationprobes, use as oligomers, or primers, for PCR, use in an array, use incomputer-readable media, use for chromosome and gene mapping, use in therecombinant production of protein, and use in generation of antisenseDNA or RNA, their chemical analogs and the like. For example, when theexpression of an mRNA is largely restricted to a particular cell ortissue type, polynucleotides of the invention can be used ashybridization probes to detect the presence of the particular cell ortissue mRNA in a sample using, e.g., in situ hybridization.

[0017] In other exemplary embodiments, the polynucleotides are used indiagnostics as expressed sequence tags for identifying expressed genesor, as well known in the art and exemplified by Vollrath et al., Science258:52-59 (1992), as expressed sequence tags for physical mapping of thehuman genome.

[0018] The polypeptides according to the invention can be used in avariety of conventional procedures and methods that are currentlyapplied to other proteins. For example, a polypeptide of the inventioncan be used to generate an antibody that specifically binds thepolypeptide. Such antibodies, particularly monoclonal antibodies, areuseful for detecting or quantitating the polypeptide in tissue. Thepolypeptides of the invention can also be used as molecular weightmarkers, and as a food supplement.

[0019] Methods are also provided for preventing, treating, orameliorating a medical condition which comprises the step ofadministering to a mammalian subject a therapeutically effective amountof a composition comprising a peptide of the present invention and apharmaceutically acceptable carrier.

[0020] In particular, the stem cell growth factor-like polypeptides andpolynucleotides of the invention may be used to induce differentiationof embryonic and adult stem cells to give rise to different cell types.They may also be used in the treatment of diseases, for example,leukemia, hemophilia, and degenerative diseases like Alzheimer'sdisease. The polynucleotides and polypeptides of the invention mayfurther be utilized to generate new tissues and organs that may aidpatients in need of transplanted tissues.

[0021] The methods of the invention also provide methods for thetreatment of disorders as recited herein which comprise theadministration of a therapeutically effective amount of a compositioncomprising a polynucleotide or polypeptide of the invention and apharmaceutically acceptable carrier to a mammalian subject exhibitingsymptoms or tendencies related to disorders as recited herein. Inaddition, the invention encompasses methods for treating diseases ordisorders as recited herein comprising the step of administering acomposition comprising compounds and other substances that modulate theoverall activity of the target gene products and a pharmaceuticallyacceptable carrier. Compounds and other substances can effect suchmodulation either on the level of target gene/protein expression ortarget protein activity. Specifically, methods are provided forpreventing, treating or ameliorating a medical condition, includingviral diseases, which comprises administering to a mammalian subject,including but not limited to humans, a therapeutically effective amountof a composition comprising a polypeptide of the invention or atherapeutically effective amount of a composition comprising a bindingpartner of (e.g., antibody specifically reactive for) stem cell growthfactor-like polypeptides of the invention. The mechanics of theparticular condition or pathology will dictate whether the polypeptidesof the invention or binding partners (or inhibitors) of these would bebeneficial to the individual in need of treatment.

[0022] According to this method, polypeptides of the invention can beadministered to produce an in vitro or in vivo inhibition of cellularfunction. A polypeptide of the invention can be administered in vivoalone or as an adjunct to other therapies. Conversely, protein or otheractive ingredients of the present invention may be included informulations of a particular agent to minimize side effects of such anagent.

[0023] The invention further provides methods for manufacturingmedicaments useful in the above-described methods.

[0024] The present invention further relates to methods for detectingthe presence of the polynucleotides or polypeptides of the invention ina sample (e.g. tissue or sample). Such methods can, for example, beutilized as part of prognostic and diagnostic evaluation of disorders asrecited herein and for the identification of subjects exhibiting apredisposition to such conditions.

[0025] The invention provides a method for detecting a polypeptide ofthe invention in a sample comprising contacting the sample with acompound that binds to and forms a complex with the polypeptide underconditions and for a period sufficient to form the complex and detectingformation of the complex, so that if a complex is formed, thepolypeptide is detected.

[0026] The invention also provides kits comprising polynucleotide probesand/or monoclonal antibodies, and optionally quantitative standards, forcarrying out methods of the invention. Furthermore, the inventionprovides methods for evaluating the efficacy of drugs, and monitoringthe progress of patients, involved in clinical trials for the treatmentof disorders as recited above.

[0027] The invention also provides methods for the identification ofcompounds that modulate (i.e., increase or decrease) the expression oractivity of the polynucleotides and/or polypeptides of the invention.Such methods can be utilized, for example, for the identification ofcompounds that can ameliorate symptoms of disorders as recited herein.Such methods can include, but are not limited to, assays for identifyingcompounds and other substances that interact with (e.g., bind to) thepolypeptides of the invention.

[0028] The invention provides a method for identifying a compound thatbinds to the polypeptide of the present invention comprising contactingthe compound with the polypeptide under conditions and for a timesufficient to form a polypeptide/compound complex and detecting thecomplex, so that if the polypeptide/compound complex is detected, acompound that binds to the polypeptide is identified.

[0029] Also provided is a method for identifying a compound that bindsto the polypeptide comprising contacting the compound with thepolypeptide in a cell for a time sufficient to form apolypeptide/compound complex wherein the complex drives expression of areporter gene sequence in the cell and detecting the complex bydetecting reporter gene sequence expression so that if thepolypeptide/compound complex is detected a compound that binds to thepolypeptide is identified.

3. BRIEF DESCRIPTION OF THE DRAWINGS

[0030]FIG. 1 shows the schematic alignment of SEQ ID NO: 24 with SEQ IDNO. 1-21,

[0031]FIG. 2 shows the BLASTX amino acid sequence alignment between stemcell growth factor-like polypeptide SEQ ID NO: 28 and tumor endothelialmarker 7 precursor protein SEQ ID NO: 36 (St. Croix et al, Science, 289,1197-1201), indicating that the two sequences share 72% similarity over441 amino acid residues and 57% identity over the same 441 amino acidresidues, wherein A=Alanine, C=Cysteine, D=Aspartic Acid, E=GlutamicAcid, F=Phenylalanine, G=Glycine, H=Histidine, I=Isoleucine, K=Lysine,L=Leucine, M=Methionine, N=Asparagine, P=Proline, Q=Glutamine,R=Arginine, S=Serine, T=Threonine, V=Valine, W=Tryptophan, Y=Tyrosine.Gaps are presented as dashes.

4. DETAILED DESCRIPTION OF THE INVENTION

[0032] The stem cell growth factor-like polypeptide of SEQ ID NO: 28 isan approximately 529-amino acid protein with a predicted molecular massof approximately 59.2-kDa unglycosylated. Protein database searches withthe BLASTP algorithm (Altschul S. F. et al., J. Mol. Evol. 36:290-300(1993) and Altschul S. F. et al., J. Mol. Biol. 21:403-10 (1990), hereinincorporated by reference) indicate that SEQ ID NO: 28 is homologous totumor endothelial marker 7 precursor protein.

[0033]FIG. 2 shows the BLASTX amino acid sequence alignment between theprotein encoded by stem cell growth factor-like polypeptide SEQ ID NO:28 and tumor endothelial marker 7 precursor protein SEQ ID NO: 36 (St.Croix et al, Science. 289, 1197-1201), indicating that the two sequencesshare 72% similarity over 441 amino acid residues and 57% identity overthe same 441 amino acid residues.

[0034] A predicted approximately thirty-residue signal peptide isencoded from approximately residue 1 through residue 30 of SEQ ID NO: 28(SEQ ID NO: 30). The extracellular portion is useful on its own. Thiscan be confirmed by expression in mammalian cells and sequencing of thecleaved product. The signal peptide region was predicted using NeuralNetwork SignalP V1.1 program (Nielsen et al, (1997) Int. J. Neur. Syst.8, 581) (from Center for Biological Sequence Analysis, The TechnicalUniversity of Denmark), and hydrophobicity analysis using theKyte/Doolittle algorithm (Kyte and Doolittle (1982) J. Mol. Biol. 157,105). One of skill in the art will recognize that the cleavage site maybe different than that predicted by the computer program. SEQ ID NO: 31is the resulting peptide when the signal peptide is removed from SEQ IDNO: 28.

[0035] A predicted approximately twenty eight-residue transmembraneregion is encoded from approximately residue 452 through residue 479 ofSEQ ID NO: 28 (SEQ ID NO: 32). It may be confirmed by expression inmammalian cells. The transmembrane region was predicted using NeuralNetwork SignalP V1.1 program (Nielsen et al, (1997) Int. J. Neur. Syst.8, 581) (from Center for Biological Sequence Analysis, The TechnicalUniversity of Denmark), and hydrophobicity analysis using theKyte/Doolittle algorithm (Kyte and Doolittle (1982) J. Mol. Biol. 157,105). One of skill in the art will recognize that the transmembraneregion may be different than that predicted by the computer program.

[0036] The stem cell growth factor-like polypeptide of SEQ ID NO: 25(identical to SEQ ID NO: 23) is an approximately 392-amino acid proteinwith a predicted molecular mass of approximately 50-kDa unglycosylated.Protein database searches with the BLASTP algorithm (Altschul S. F. etal., J. Mol. Evol. 36:290-300 (1993) and Altschul S. F. et al., J. Mol.Biol. 21:403-10 (1990), herein incorporated by reference) indicate thatSEQ ID NO: 25 is homologous to tumor endothelial marker 7 precursorprotein.

[0037] A predicted approximately twenty eight-residue transmembraneregion is encoded from approximately residue 315 through residue 342 ofSEQ ID NO: 25 (SEQ ID NO: 32). It may be confirmed by expression inmammalian cells. The transmembrane region was predicted using NeuralNetwork SignalP V1.1 program (Nielsen et al, (1997) Int. J. Neur. Syst.8, 581) (from Center for Biological Sequence Analysis, The TechnicalUniversity of Denmark), and hydrophobicity analysis using theKyte/Doolittle algorithm (Kyte and Doolittle (1982) J. Mol. Biol. 157,105). One of skill in the art will recognize that the transmembraneregion may be different than that predicted by the computer program.

[0038] In particular, the stem cell growth factor-like polypeptides andpolynucleotides of the invention may be used to induce differentiationof embryonic and adult stem cells to give rise to different cell types.They may also be used in the treatment of leukemia, hemophilia, anddegenerative diseases like Alzheimer's disease. The polynucleotides andpolypeptides of the invention may further be utilized to generate newtissues and organs that may aid patients in need of transplantedtissues.

4.1 Definitions

[0039] It must be noted that as used herein and in the appended claims,the singular forms “a”, “an” and “the” include plural references unlessthe context clearly dictates otherwise.

[0040] The term “active” refers to those forms of the polypeptide thatretain the biologic and/or immunologic activities of any naturallyoccurring polypeptide. According to the invention, the terms“biologically active” or “biological activity” refer to a protein orpeptide having structural, regulatory or biochemical functions of anaturally occurring molecule. Likewise “biologically active” or“biological activity” refers to the capability of the natural,recombinant or synthetic stem cell growth factor-like peptide, or anypeptide thereof, to induce a specific biological response in appropriateanimals or cells and to bind with specific antibodies. The term “stemcell growth factor-like biological activity” refers to biologicalactivity that is similar to the biological activity of a stem cellgrowth factor-like.

[0041] The term “activated cells” as used in this application are thosecells which are engaged in extracellular or intracellular membranetrafficking, including the export of secretory or enzymatic molecules aspart of a normal or disease process.

[0042] The terms “complementary” or “complementarity” refer to thenatural binding of polynucleotides by base pairing. For example, thesequence 5′-AGT-3′ binds to the complementary sequence 3′-TCA-5′.Complementarity between two single-stranded molecules may be “partial”such that only some of the nucleic acids bind or it may be “complete”such that total complementarity exists between the single strandedmolecules. The degree of complementarity between the nucleic acidstrands has significant effects on the efficiency and strength of thehybridization between the nucleic acid strands.

[0043] The term “embryonic stem cells (ES)” refers to a cell that cangive rise to many differentiated cell types in an embryo or an adult,including the germ cells. The term “germ line stem cells (GSCs)” refersto stem cells derived from primordial stem cells that provide a steadyand continuous source of germ cells for the production of gametes. Theterm “primordial germ cells (PGCs)” refers to a small population ofcells set aside from other cell lineages particularly from the yolk sac,mesenteries, or gonadal ridges during embryogenesis that have thepotential to differentiate into germ cells and other cells. PGCs are thesource from which GSCs and ES cells are derived The PGCs, the GSCs andthe ES cells are capable of self-renewal. Thus these cells not onlypopulate the germ line and give rise to a plurality of terminallydifferentiated cells that comprise the adult specialized organs, but areable to regenerate themselves. The term “totipotent” refers to thecapability of a cell to differentiate into all of the cell types of anadult organism. The term “pluripotent” refers to the capability of acell to differentiate into a number of differentiated cell types thatare present in an adult organism. A pluripotent cell is restricted inits differentiation capability in comparison to a totipotent cell.

[0044] The term “expression modulating fragment,” EMF, means a series ofnucleotides that modulates the expression of an operable linked ORF oranother EMF.

[0045] As used herein, a sequence is said to “modulate the expression ofan operably linked sequence” when the expression of the sequence isaltered by the presence of the EMF. EMFs include, but are not limitedto, promoters, and promoter modulating sequences (inducible elements).One class of EMFs is nucleic acid fragments which induce the expressionof an operably linked ORF in response to a specific regulatory factor orphysiological event.

[0046] The terms “nucleotide sequence” or “nucleic acid” or“polynucleotide ” or “oligonucleotide” are used interchangeably andrefer to a heteropolymer of nucleotides or the sequence of thesenucleotides. These phrases also refer to DNA or RNA of genomic orsynthetic origin which may be single-stranded or double-stranded and mayrepresent the sense or the antisense strand, to peptide nucleic acid(PNA) or to any DNA-like or RNA-like material. In the sequences, A isadenine, C is cytosine, G is guanine, and T is thymine, while N is A, T,G, or C. It is contemplated that where the polynucleotide is RNA, the T(thymine) in the sequence herein may be replaced with U (uracil).Generally, nucleic acid segments provided by this invention may beassembled from fragments of the genome and short oligonucleotidelinkers, or from a series of oligonucleotides, or from individualnucleotides, to provide a synthetic nucleic acid which is capable ofbeing expressed in a recombinant transcriptional unit comprisingregulatory elements derived from a microbial or viral operon, or aeukaryotic gene.

[0047] The terms “oligonucleotide fragment” or a “polynucleotidefragment”, “portion,” or “segment” or “probe” or “primer” are usedinterchangeably and refer to a sequence of nucleotide residues which areat least about 5 nucleotides, more preferably at least about 7nucleotides. more preferably at least about 9 nucleotides, morepreferably at least about 11 nucleotides and most preferably at leastabout 17 nucleotides. The fragment is preferably less than about 500nucleotides, preferably less than about 200 nucleotides, more preferablyless than about 100 nucleotides, more preferably less than about 50nucleotides and most preferably less than 30 nucleotides. Preferably theprobe is from about 6 nucleotides to about 200 nucleotides, preferablyfrom about 15 to about 50 nucleotides, more preferably from about 17 to30 nucleotides and most preferably from about 20 to 25 nucleotides.Preferably the fragments can be used in polymerase chain reaction (PCR),various hybridization procedures or mocroarray procedures to identify oramplify identical or related parts of mRNA or DNA molecules. A fragmentor segment may uniquely identify each polynucleotide sequence of thepresent invention. Preferably the fragment comprises a sequencesubstantially similar to a portion of SEQ ID NO: 1-22, 24, 26-27, 29, or33.

[0048] Probes may, for example, be used to determine whether specificmRNA molecules are present in a cell or tissue or to isolate similarnucleic acid sequences from chromosomal DNA as described by Walsh et al,(Walsh. P. S. et al., 1992, PCR Methods Appl 1:241-250). They may belabeled by nick translation, Klenow fill-in reaction, PCR, or othermethods well known in the art. Probes of the present invention, theirpreparation and/or labeling are elaborated in Sambrook, J. et al., 1989,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,NY; or Ausubel, F. M. et al., 1989, Current Protocols in MolecularBiology, John Wiley & Sons, New York N.Y., both of which areincorporated herein by reference in their entirety.

[0049] The nucleic acid sequences of the present invention also includethe sequence information from any of the nucleic acid sequences of SEQID NO: 1-22, 24, 26-27, 29, or 33. The sequence information can be asegment of SEQ ID NO: 1-22, 24, 26-27, 29, or 33 that uniquelyidentifies or represents the sequence information of SEQ ID NO: 1-22,24, 26-27, 29, or 33. One such segment can be a twenty-mer nucleic acidsequence because the probability that a twenty-mer is fully matched inthe human genome is 1 in 300. In the human genome, there are threebillion base pairs in one set of chromosomes. Because 4²⁰ possibletwenty-mers exist, there are 300 times more twenty-mers than there arebase pairs in a set of human chromosomes. Using the same analysis, theprobability for a seventeen-mer to be fully matched in the human genomeis approximately 1 in 5. When these segments are used in arrays forexpression studies, fifteen-mer segments can be used. The probabilitythat the fifteen-mer is fully matched in the expressed sequences is alsoapproximately one in five because expressed sequences comprise less thanapproximately 5% of the entire genome sequence.

[0050] Similarly, when using sequence information for detecting a singlemismatch, a segment can be a twenty-five mer. The probability that thetwenty-five mer would appear in a human genome with a single mismatch iscalculated by multiplying the probability for a full match (1÷4²⁵) timesthe increased probability for mismatch at each nucleotide position(3×25). The probability that an eighteen mer with a single mismatch canbe detected in an array for expression studies is approximately one infive. The probability that a twenty-mer with a single mismatch can bedetected in a human genome is approximately one in five.

[0051] The term “open reading frame,” ORF, means a series of nucleotidetriplets coding for amino acids without any termination codons and is asequence translatable into protein.

[0052] The terms “operably linked” or “operably associated” refer tofunctionally related nucleic acid sequences. For example, a promoter isoperably associated or operably linked with a coding sequence if thepromoter controls the transcription of the coding sequence. Whileoperably linked nucleic acid sequences can be contiguous and in the samereading frame, certain genetic elements e.g. repressor genes are notcontiguously linked to the coding sequence but still controltranscription/translation of the coding sequence.

[0053] The term “pluripotent” refers to the capability of a cell todifferentiate into a number of differentiated cell types that arepresent in an adult organism. A pluripotent cell is restricted in itsdifferentiation capability in comparison to a totipotent cell.

[0054] The terms “polypeptide” or “peptide” or “amino acid sequence”refer to an oligopeptide. peptide, polypeptide or protein sequence orfragment thereof and to naturally occurring or synthetic molecules. Apolypeptide “fragment,” “portion,” or “segment” is a stretch of aminoacid residues of at least about 5 amino acids, preferably at least about7 amino acids, more preferably at least about 9 amino acids and mostpreferably at least about 17 or more amino acids. The peptide preferablyis not greater than about 200 amino acids, more preferably less than 150amino acids and most preferably less than 100 amino acids. Preferablythe peptide is from about 5 to about 200 amino acids. To be active, anypolypeptide must have sufficient length to display biological and/orimmunological activity.

[0055] The term “naturally occurring polypeptide” refers to polypeptidesproduced by cells that have not been genetically engineered andspecifically contemplates various polypeptides arising frompost-translational modifications of the polypeptide including, but notlimited to, acetylation, carboxylation, glycosylation, phosphorylation,lipidation and acylation.

[0056] The term “translated protein coding portion” means a sequencewhich encodes for the full length protein which may include any leadersequence or a processing sequence.

[0057] The term “mature protein coding sequence” refers to a sequencewhich encodes a peptide or protein without any leader/signal sequence.The “mature protein portion” refers to that portion of the proteinwithout the leader/signal sequence. The peptide may have the leadersequences removed during processing in the cell or the protein may havebeen produced synthetically or using a polynucleotide only encoding forthe mature protein coding sequence. It is contemplated that the matureprotein portion may or may not include an initial methionine residue.The initial methionine is often removed during processing of thepeptide.

[0058] The term “derivative” refers to polypeptides chemically modifiedby such techniques as ubiquitination, labeling (e.g., with radionuclidesor various enzymes), covalent polymer attachment such as pegylation(derivatization with polyethylene glycol) and insertion or substitutionby chemical synthesis of amino acids such as ornithine, which do notnormally occur in human proteins.

[0059] The term “variant” (or “analog”) refers to any polypeptidediffering from naturally occurring polypeptides by amino acidinsertions, deletions, and substitutions, created using, e g.,recombinant DNA techniques. Guidance in determining which amino acidresidues may be replaced, added or deleted without abolishing activitiesof interest, may be found by comparing the sequence of the particularpolypeptide with that of homologous peptides and minimizing the numberof amino acid sequence changes made in regions of high homology(conserved regions) or by replacing amino acids with consensus sequence.

[0060] Alternatively, recombinant variants encoding these same orsimilar polypeptides may be synthesized or selected by making use of the“redundancy” in the genetic code. Various codon substitutions, such asthe silent changes which produce various restriction sites, may beintroduced to optimize cloning into a plasmid or viral vector orexpression in a particular prokaryotic or eukaryotic system. Mutationsin the polynucleotide sequence may be reflected in the polypeptide ordomains of other peptides added to the polypeptide to modify theproperties of any part of the polypeptide, to change characteristicssuch as ligand-binding affinities, interchain affinities, ordegradation/turnover rate.

[0061] Preferably, amino acid “substitutions” are the result ofreplacing one amino acid with another amino acid having similarstructural and/or chemical properties, i.e., conservative amino acidreplacements. “Conservative” amino acid substitutions may be made on thebasis of similarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues involved.For example, nonpolar (hydrophobic) amino acids include alanine,leucine, isoleucine, valine, proline, phenylalanine, tryptophan, andmethionine; polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine; positivelycharged (basic) amino acids include arginine, lysine, and histidine; andnegatively charged (acidic) amino acids include aspartic acid andglutamic acid. “Insertions” or “deletions” are preferably in the rangeof about 1 to 20 amino acids, more preferably 1 to 10 amino acids. Thevariation allowed may be experimentally determined by systematicallymaking insertions, deletions, or substitutions of amino acids in apolypeptide molecule using recombinant DNA techniques and assaying theresulting recombinant variants for activity.

[0062] Alternatively, where alteration of function is desired,insertions, deletions or non-conservative alterations can be engineeredto produce altered polypeptides. Such alterations can, for example,alter one or more of the biological functions or biochemicalcharacteristics of the polypeptides of the invention. For example, suchalterations may change polypeptide characteristics such asligand-binding affinities, interchain affinities, ordegradation/turnover rate. Further, such alterations can be selected soas to generate polypeptides that are better suited for expression, scaleup and the like in the host cells chosen for expression. For example,cysteine residues can be deleted or substituted with another amino acidresidue in order to eliminate disulfide bridges.

[0063] The terms “purified” or “substantially purified” as used hereindenotes that the indicated nucleic acid or polypeptide is present in thesubstantial absence of other biological macromolecules, e.g.,polynucleotides, proteins, and the like. In one embodiment, thepolynucleotide or polypeptide is purified such that it constitutes atleast 95% by weight, more preferably at least 99% by weight, of theindicated biological macromolecules present (but water, buffers, andother small molecules, especially molecules having a molecular weight ofless than 1000 daltons, can be present).

[0064] The term “isolated” as used herein refers to a nucleic acid orpolypeptide separated from at least one other component (e.g., nucleicacid or polypeptide) present with the nucleic acid or polypeptide in itsnatural source. In one embodiment, the nucleic acid or polypeptide isfound in the presence of (if anything) only a solvent, buffer, ion, orother components normally present in a solution of the same. The terms“isolated” and “purified” do not encompass nucleic acids or polypeptidespresent in their natural source.

[0065] The term “recombinant,” when used herein to refer to apolypeptide or protein, means that a polypeptide or protein is derivedfrom recombinant (e.g., microbial, insect, or mammalian) expressionsystems. “Microbial” refers to recombinant polypeptides or proteins madein bacterial or fungal (e.g., yeast) expression systems. As a product,“recombinant microbial” defines a polypeptide or protein essentiallyfree of native endogenous substances and unaccompanied by associatednative glycosylation. Polypeptides or proteins expressed in mostbacterial cultures, e.g., E. coli, will be free of glycosylationmodifications; polypeptides or proteins expressed in yeast will have aglycosylation pattern in general different from those expressed inmammalian cells.

[0066] The term “recombinant expression vehicle or vector” refers to aplasmid or phage or virus or vector, for expressing a polypeptide from aDNA (RNA) sequence. An expression vehicle can comprise a transcriptionalunit comprising an assembly of (1) a genetic element or elements havinga regulatory role in gene expression, for example, promoters orenhancers, (2) a structural or coding sequence which is transcribed intomRNA and translated into protein, and (3) appropriate transcriptioninitiation and termination sequences. Structural units intended for usein yeast or eukaryotic expression systems preferably include a leadersequence enabling extracellular secretion of translated protein by ahost cell. Alternatively, where recombinant protein is expressed withouta leader or transport sequence, it may include an amino terminalmethionine residue. This residue may or may not be subsequently cleavedfrom the expressed recombinant protein to provide a final product.

[0067] The term “recombinant expression system” means host cells whichhave stably integrated a recombinant transcriptional unit intochromosomal DNA or carry the recombinant transcriptional unitextrachromosomally. Recombinant expression systems as defined hereinwill express heterologous polypeptides or proteins upon induction of theregulatory elements linked to the DNA segment or synthetic gene to beexpressed. This term also means host cells which have stably integrateda recombinant genetic element or elements having a regulatory role ingene expression, for example, promoters or enhancers. Recombinantexpression systems as defined herein will express polypeptides orproteins endogenous to the cell upon induction of the regulatoryelements linked to the endogenous DNA seament or gene to be expressed.The cells can be prokaryotic or eukaryotic.

[0068] The term “secreted” includes a protein that is transported acrossor through a membrane, including transport as a result of signalsequences in its amino acid sequence when it is expressed in a suitablehost cell. “Secreted” proteins include without limitation proteinssecreted wholly (e.g., soluble proteins) or partially (e.g., receptors)from the cell in which they are expressed. “Secreted” proteins alsoinclude without limitation proteins that are transported across themembrane of the endoplasmic reticulum. “Secreted” proteins are alsointended to include proteins containing non-typical signal sequences(e.g. Interleukin-1 Beta, see Krasney, P. A. and Young, P. R. (1992)Cytokine 4(2):134-143) and factors released from damaged cells (e.g.Interleukin-1 Receptor Antagonist, see Arend, W. P. et. al, (1998) Annu.Rev. Immunol. 16:27-55)

[0069] Where desired, an expression vector may be designed to contain a“signal or leader sequence” which will direct the polypeptide throughthe membrane of a cell. Such a sequence may be naturally present on thepolypeptides of the present invention or provided from heterologousprotein sources by recombinant DNA techniques.

[0070] The term “stringent” is used to refer to conditions that arecommonly understood in the art as stringent. Stringent conditions caninclude highly stringent conditions (i.e., hybridization to filter-boundDNA in 0.5 M NaHPO₄, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65°C. and washing in 0.1×SSC/0.1% SDS at 68° C.), and moderately stringentconditions (i.e., washing in 0.2×SSC/0.1% SDS at 42° C.). Otherexemplary hybridization conditions are described herein in the examples.

[0071] In instances of hybridization of deoxyoligonucleotides,additional exemplary stringent hybridization conditions include washingin 6×SSC/0.05% sodium pyrophosphate at 37° C. (for 14-baseoligonucleotides), 48° C. (for 17-base oligonucleotides), 55° C. (for20-base oligonucleotides), and 60° C. (for 23-base oligonucleotides).

[0072] As used herein, “substantially equivalent” can refer both tonucleotide and amino acid sequences, for example a mutant sequence, thatvaries from a reference sequence by one or more substitutions,deletions, or additions, the net effect of which does not result in anadverse functional dissimilarity between the reference and subjectsequences. Typically, such a substantially equivalent sequence variesfrom one of those listed herein by no more than about 35% (i.e., thenumber of individual residue substitutions, additions, and/or deletionsin a substantially equivalent sequence, as compared to the correspondingreference sequence, divided by the total number of residues in thesubstantially equivalent sequence is about 0.35 or less). Such asequence is said to have 65% sequence identity to the listed sequence.In one embodiment, a substantially equivalent, e.g., mutant, sequence ofthe invention varies from a listed sequence by no more than 30% (70%sequence identity); in a variation of this embodiment, by no more than25% (75% sequence identity); and in a further variation of thisembodiment, by no more than 20% (80% sequence identity) and in a furthervariation of this embodiment, by no more than 10% (90% sequenceidentity) and in a further variation of this embodiment, by no more that5% (95% sequence identity). Substantially equivalent, e.g., mutant,amino acid sequences according to the invention preferably have at least80% sequence identity with a listed amino acid sequence, more preferablyat least 90% sequence identity. Substantially equivalent nucleotidesequence of the invention can have lower percent sequence identities,taking into account, for example, the redundancy or degeneracy of thegenetic code. Preferably, nucleotide sequence has at least about 65%identity, more preferably at least about 75% identity, and mostpreferably at least about 95% identity. For the purposes of the presentinvention, sequences having substantially equivalent biological activityand substantially equivalent expression characteristics are consideredsubstantially equivalent. For the purposes of determining equivalence,truncation of the mature sequence (e.g., via a mutation which creates aspurious stop codon) should be disregarded. Sequence identity may bedetermined, e.g., using the Jotun Hein method (Hein. J. (1990) MethodsEnzymol. 183:626-645). Identity between sequences can also be determinedby other methods known in the art, e.g. by varying hybridizationconditions.

[0073] The term “totipotent” refers to the capability of a cell todifferentiate into all of the cell types of an adult organism.

[0074] The term “transformation” means introducing DNA into a suitablehost cell so that the DNA is replicable, either as an extrachromosomalelement, or by chromosomal integration. The term “transfection” refersto the taking up of an expression vector by a suitable host cell,whether or not any coding sequences are in fact expressed. The term“infection” refers to the introduction of nucleic acids into a suitablehost cell by use of a virus or viral vector.

[0075] As used herein, an “uptake modulating fragment,” UMF, means aseries of nucleotides which mediate the uptake of a linked DNA fragmentinto a cell. UMFs can be readily identified using known UMFs as a targetsequence or target motif with the computer-based systems describedbelow. The presence and activity of a UMF can be confirmed by attachingthe suspected UMF to a marker sequence. The resulting nucleic acidmolecule is then incubated with an appropriate host under appropriateconditions and the uptake of the marker sequence is determined. Asdescribed above, a UMF will increase the frequency of uptake of a linkedmarker sequence.

[0076] Each of the above terms is meant to encompass all that isdescribed for each, unless the context dictates otherwise.

4.2 Nucleic Acids of the Invention

[0077] The invention is based on the discovery of a novel stem cellgrowth factor-like polypeptide, the polynucleotides encoding the stemcell growth factor-like polypeptide and the use of these compositionsfor the diagnosis, treatment or prevention of cancers and otherimmunological disorders.

[0078] The isolated polynucleotides of the invention include, but arenot limited to a polynucleotide comprising any of the nucleotidesequences of SEQ ID NO: 1-22, 24, 26-27, 29, or 33; a fragment of SEQ IDNO: 1-22, 24, 26-27, 29, or 33; a polynucleotide comprising the fulllength protein coding sequence of SEQ ID NO: 1-22, 24, 26-27, 29, or 33(for example coding for SEQ ID NO: 23, 25, or 28); and a polynucleotidecomprising the nucleotide sequence encoding the mature protein codingsequence of the polynucleotides of any one of SEQ ID NO: 1-22, 24,26-27, 29, or 33. The polynucleotides of the present invention alsoinclude, but are not limited to, a polynucleotide that hybridizes understringent conditions to (a) the complement of any of the nucleotidessequences of SEQ ID NO: 1-22, 24, 26-27, 29, or 33; (b) a polynucleotideencoding any one of the polypeptides of SEQ ID NO: 23, 25, 28, 30-32,34, or 35; (c) a polynucleotide which is an allelic variant of anypolynucleotides recited above; (d) a polynucleotide which encodes aspecies homolog of any of the proteins recited above; or (e) apolynucleotide that encodes a polypeptide comprising a specific domainor truncation of the polypeptides of SEQ ID NO: 23, 25, 28, 30-32, 34,or 35. Domains of interest may depend on the nature of the encodedpolypeptide; e.g., domains in receptor-like polypeptides includeligand-binding, extracellular, transmembrane, or cytoplasmic domains, orcombinations thereof; domains in immunoglobulin-like proteins includethe variable immunoglobulin-like domains; domains in enzyme-likepolypeptides include cataletic and substrate binding domains; anddomains in ligand polypeptides include receptor-bindine domains.

[0079] The polynucleotides of the invention include naturally occurringor wholly or partially synthetic DNA, e.g., cDNA and genomic DNA, andRNA, e.g., mRNA. The polynucleotides may include all of the codingregion of the cDNA or may represent a portion of the coding region ofthe cDNA.

[0080] The present invention also provides genes corresponding to thecDNA sequences disclosed herein. The corresponding genes can be isolatedin accordance with known methods using the sequence informationdisclosed herein. Such methods include the preparation of probes orprimers from the disclosed sequence information for identificationand/or amplification of genes in appropriate genomic libraries or othersources of genomic materials. Further 5′ and 3′ sequence can be obtainedusing methods known in the art. For example, full length cDNA or genomicDNA that corresponds to any of the polynucleotides of SEQ ID NO: 1-22,24, 26-27, 29, or 33 can be obtained by screening appropriate cDNA orgenomic DNA libraries under suitable hybridization conditions using anyof the polynucleotides of SEQ ID NO: 1-22, 24, 26-27, 29, or 33 or aportion thereof as a probe. Alternatively, the polynucleotides of SEQ IDNO: 1-22, 24, 26-27, 29, or 33 may be used as the basis for suitableprimer(s) that allow identification and/or amplification of genes inappropriate genomic DNA or cDNA libraries.

[0081] The nucleic acid sequences of the invention can be assembled fromESTs and sequences (including cDNA and genomic sequences) obtained fromone or more public databases, such as dbEST, gbpri, and UniGene. The ESTsequences can provide identifying sequence information, representativefragment or segment information, or novel segment information for thefull-length gene.

[0082] The polynucleotides of the invention also provide polynucleotidesincluding nucleotide sequences that are substantially equivalent to thepolynucleotides recited above. Polynucleotides according to theinvention can have, e.g., at least about 65%, at least about 70%, atleast about 75%, at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, or 89%, more at least about 90%, 91%, 92%, 93%, or 94% and evenmore typically at least about 95%, 96%, 97%, 98% or 99% sequenceidentity to a polynucleotide recited above.

[0083] Included within the scope of the nucleic acid sequences of theinvention are nucleic acid sequence fragments that hybridize understringent conditions to any of the nucleotide sequences of SEQ ID NO:1-22, 24, 26-27, 29, or 33, or complements thereof, which fragment isgreater than about 5 nucleotides, preferably 7 nucleotides, morepreferably greater than 9 nucleotides and most preferably greater than17 nucleotides. Fragments of, e.g. 15, 17, or 20 nucleotides or morethat are selective for (i.e, specifically hybridize to any one of thepolynucleotides of the invention) are contemplated. Probes capable ofspecifically hybridizing to a polynucleotide can differentiatepolynucleotide sequences of the invention from other polynucleotidesequences in the same family of genes or can differentiate human genesfrom genes of other species, and are preferably based on uniquenucleotide sequences.

[0084] The sequences falling within the scope of the present inventionare not limited to these specific sequences, but also include allelicand species variations thereof. Allelic and species variations can beroutinely determined by comparing the sequence provided in SEQ ID NO:1-22, 24, 26-27, 29, or 33, a representative fragment thereof, or anucleotide sequence at least 90% identical, preferably 95% identical, toSEQ ID NO: 1-22, 24, 26-27, 29, or 33 with a sequence from anotherisolate of the same species. Furthermore, to accommodate codonvariability, the invention includes nucleic acid molecules coding forthe same amino acid sequences as do the specific ORFs disclosed herein.In other words, in the coding region of an ORF, substitution of onecodon for another codon that encodes the same amino acid is expresslycontemplated.

[0085] The nearest neighbor result for the nucleic acids of the presentinvention, including SEQ ID NO: 1-22, 24, 26-27, 29, or 33, can beobtained by searching a database using an algorithm or a program.Preferably, a BLAST which stands for Basic Local Alignment Search Toolis used to search for local sequence alignments (Altschul, S. F. J Mol.Evol. 36 290-300 (1993) and Altschul S. F, et al, J. Mol. Biol.21:403-410 (1990))

[0086] Species homologs (or orthologs) of the disclosed polynucleotidesand proteins are also provided by the present invention. Specieshomologs may be isolated and identified by making suitable probes orprimers from the sequences provided herein and screening a suitablenucleic acid source from the desired species.

[0087] The invention also encompasses allelic variants of the disclosedpolynucleotides or proteins; that is, naturally-occurring alternativeforms of the isolated polynucleotide which also encode proteins whichare identical, homologous or related to that encoded by thepolynucleotides.

[0088] The nucleic acid sequences of the invention are further directedto sequences which encode variants of the described nucleic acids. Theseamino acid sequence variants may be prepared by methods known in the artby introducing appropriate nucleotide chances into a native or variantpolynucleotide. There are two variables in the construction of aminoacid sequence variants: the location of the mutation and the nature ofthe mutation. Nucleic acids encoding the amino acid sequence variantsare preferably constructed by mutating the polynucleotide to encode anamino acid sequence that does not occur in nature. These nucleic acidalterations can be made at sites that differ in the nucleic acids fromdifferent species (variable positions) or in highly conserved regions(constant regions). Sites at such locations will typically be modifiedin series, e.g., by substituting first with conservative choices (e.g.,hydrophobic amino acid to a different hydrophobic amino acid) and thenwith more distant choices (e.g., hydrophobic amino acid to a chargedamino acid), and then deletions or insertions may be made at the targetsite. Amino acid sequence deletions generally range from about 1 to 30residues, preferably about 1 to 10 residues, and are typicallycontiguous. Amino acid insertions include amino- and/orcarboxyl-terminal fusions ranging in length from one to one hundred ormore residues, as well as intrasequence insertions of single or multipleamino acid residues. Intrasequence insertions may range generally fromabout 1 to 10 amino residues, preferably from 1 to 5 residues. Examplesof terminal insertions include the heterologous signal sequencesnecessary for secretion or for intracellular targeting in different hostcells and sequences such as FLAG or poly-histidine sequences useful forpurifying the expressed protein.

[0089] In a preferred method, polynucleotides encoding the novel aminoacid sequences are changed via site-directed mutagenesis. This methoduses oligonucleotide sequences to alter a polynucleotide to encode thedesired amino acid variant, as well as sufficient adjacent nucleotideson both sides of the changed amino acid to form a stable duplex oneither side of the site being changed. In general, the techniques ofsite-directed mutagenesis are well known to those of skill in the artand this technique is exemplified by publications such as, Edelman etal., DNA 2:183 (1983). A versatile and efficient method for producingsite-specific changes in a polynucleotide sequence was published byZoller and Smith, Nucleic Acids Res. 10:6487-6500 (1982). PCR may alsobe used to create amino acid sequence variants of the novel nucleicacids. When small amounts of template DNA are used as starting material,primer(s) that differs slightly in sequence from the correspondingregion in the template DNA can generate the desired amino acid variant.PCR amplification results in a population of product DNA fragments thatdiffer from the polynucleotide template encoding the polypeptide at theposition specified by the primer. The product DNA fragments replace thecorresponding region in the plasmid and this gives a polynucleotideencoding the desired amino acid variant.

[0090] A further technique for generating amino acid variants is thecassette mutagenesis technique described in Wells et al., Gene 34:315(1985); and other mutagenesis techniques well known in the art, such as,for example, the techniques in Sambrook et al., supra, and CurrentProtocols in Molecular Biology, Ausubel et al. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be used in the practice of the invention for the cloning andexpression of these novel nucleic acids. Such DNA sequences includethose which are capable of hybridizing to the appropriate novel nucleicacid sequence under stringent conditions.

[0091] Polynucleotides encoding preferred polypeptide truncations of theinvention can be used to generate polynucleotides encoding chimeric orfusion proteins comprising one or more domains of the invention andheterologous protein sequences.

[0092] The polynucleotides of the invention additionally include thecomplement of any of the polynucleotides recited above. Thepolynucleotide can be DNA (genomic, cDNA, amplified, or synthetic) orRNA. Methods and algorithms for obtaining such polynucleotides are wellknown to those of skill in the art and can include, for example, methodsfor determining hybridization conditions that can routinely isolatepolynucleotides of the desired sequence identities.

[0093] In accordance with the invention, polynucleotide sequencescomprising the mature protein coding sequences, coding for any one ofSEQ ID NO: 23, 25, 28, 30-32, 34, or 35, or functional equivalentsthereof, may be used to generate recombinant DNA molecules that directthe expression of that nucleic acid, or a functional equivalent thereof,in appropriate host cells. Also included are the cDNA inserts of any ofthe clones identified herein.

[0094] A polynucleotide according to the invention can be joined to anyof a variety of other nucleotide sequences by well-establishedrecombinant DNA techniques (see Sambrook J et al, (1989) MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY). Usefulnucleotide sequences for joining to polynucleotides include anassortment of vectors, e.g., plasmids, cosmids, lambda phagederivatives, phagemids, and the like, that are well known in the art.Accordingly, the invention also provides a vector including apolynucleotide of the invention and a host cell containing thepolynucleotide. In general, the vector contains an origin of replicationfunctional in at least one organism, convenient restriction endonucleasesites, and a selectable marker for the host cell. Vectors according tothe invention include expression vectors, replication vectors, probegeneration vectors, and sequencing vectors. A host cell according to theinvention can be a prokaryotic or eukaryotic cell and can be aunicellular organism or part of a multicellular organism.

[0095] The present invention further provides recombinant constructscomprising a nucleic acid having any of the nucleotide sequences of SEQID NO: 1-22, 24, 26-27, 29, or 33 or a fragment thereof or any otherpolynucleotides of the invention. In one embodiment, the recombinantconstructs of the present invention comprise a vector, such as a plasmidor viral vector, into which a nucleic acid having any of the nucleotidesequences of SEQ ID NO: 1-22, 24, 26-27, 29, or 33 or a fragment thereofis inserted, in a forward or reverse orientation. In the case of avector comprising one of the ORFs of the present invention, the vectormay further comprise regulator sequences, including for example, apromoter, operably linked to the ORF. Large numbers of suitable vectorsand promoters are known to those of skill in the art and arecommercially available for generating the recombinant constructs of thepresent invention. The following vectors are provided by way of example.Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a,pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3,pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLneo, pSV2cat, pOG44, PXTI, pSG(Stratagene) pSVK3, pBPV, pMSG, and pSVL (Pharmacia).

[0096] The isolated polynucleotide of the invention may be operablylinked to an expression control sequence such as the pMT2 or pEDexpression vectors disclosed in Kaufman et al., Nucleic Acids Res. 19,4485-4490 (1991), in order to produce the protein recombinantly. Manysuitable expression control sequences are known in the art. Generalmethods of expressing recombinant proteins are also known and areexemplified in R. Kaufman, Methods in Enzymology 185, 537-566 (1990). Asdefined herein “operably linked” means that the isolated polynucleotideof the invention and an expression control sequence are situated withina vector or cell in such a way that the protein is expressed by a hostcell which has been transformed (transfected) with the ligatedpolynucleotide/expression control sequence.

[0097] Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include lacI, lacZ, T3, T7, gpt, lambda PR, and trc.Eukaryotic promoters include CMV immediate early, HSV thymidine kinase,early and late SV40, LTRs from retrovirus, and mouse metallothionein-I.Selection of the appropriate vector and promoter is well within thelevel of ordinary skill in the art. Generally, recombinant expressionvectors will include origins of replication and selectable markerspermitting transformation of the host cell, e.g., the ampicillinresistance gene of E. coli and S. cerevisiae TRP1 gene, and a promoterderived from a highly expressed gene to direct transcription of adownstream structural sequence. Such promoters can be derived fromoperons encoding glycolytic enzymes such as 3-phosphoglycerate kinase(PGK), a-factor, acid phosphatase, or heat shock proteins, among others.The heterologous structural sequence is assembled in appropriate phasewith translation initiation and termination sequences, and preferably, aleader sequence capable of directing secretion of translated proteininto the periplasmic space or extracellular medium. Optionally, theheterologous sequence can encode a fusion protein including an aminoterminal identification peptide imparting desired characteristics, e.g.,stabilization or simplified purification of expressed recombinantproduct. Useful expression vectors for bacterial use are constructed byinserting a structural DNA sequence encoding a desired protein togetherwith suitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

[0098] As a representative but non-limiting example, useful expressionvectors for bacterial use can comprise a selectable marker and bacterialorigin of replication derived from commercially available plasmidscomprising genetic elements of the well known cloning vector pBR322(ATCC 37017). Such commercial vectors include, for example, pKK223-3(Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM 1 (Promega Biotech,Madison, Wis. USA). These pBR322 “backbone” sections are combined withan appropriate promoter and the structural sequence to be expressed.Following transformation of a suitable host strain and growth of thehost strain to an appropriate cell density, the selected promoter isinduced or derepressed by appropriate means (e.g., temperature shift orchemical induction) and cells are cultured for an additional period.Cells are typically harvested by centrifugation, disrupted by physicalor chemical means, and the resulting crude extract retained for furtherpurification.

[0099] Polynucleotides of the invention can also be used to induceimmune responses. For example, as described in Fan et al., Nat. Biotech.17:870-872 (1999), incorporated herein by reference, nucleic acidsequences encoding a polypeptide may be used to generate antibodiesagainst the encoded polypeptide following topical administration ofnaked plasmid DNA or following injection, and preferably intramuscularinjection of the DNA. The nucleic acid sequences are preferably insertedin a recombinant expression vector and may be in the form of naked DNA.

[0100] 4.2.1 Antisense Nucleic Acids

[0101] Another aspect of the invention pertains to isolated antisensenucleic acid molecules that can hybridize to, or are complementary to,the nucleic acid molecule comprising the stem cell growth factor-likenucleotide sequence, or fragments, analogs or derivatives thereof. An“antisense” nucleic acid comprises a nucleotide sequence that iscomplementary to a “sense” nucleic acid encoding a protein (e.g.,complementary to the coding strand of a double-stranded cDNA molecule orcomplementary to an mRNA sequence). In specific aspects, antisensenucleic acid molecules are provided that comprise a sequencecomplementary to at least about 10, 25, 50, 100, 250 or 500 nucleotidesor an entire stem cell growth factor-like coding strand, or to only aportion thereof. Nucleic acid molecules encoding fragments, homologs,derivatives and analogs of a stem cell growth factor-like or antisensenucleic acids complementary to a stem cell growth factor-like nucleicacid sequence of are additionally provided.

[0102] In one embodiment, an antisense nucleic acid molecule isantisense to a “coding region” of the coding strand of a nucleotidesequence encoding a stem cell growth factor-like protein. The term“coding region” refers to the region of the nucleotide sequencecomprising codons which are translated into amino acid residues. Inanother embodiment, the antisense nucleic acid molecule is antisense toa “conceding region” of the coding strand of a nucleotide sequenceencoding the stem cell growth factor-like protein. The term “concedingregion” refers to 5′ and 3′ sequences which flank the coding region thatare not translated into amino acids (i.e., also referred to as 5′ and 3′untranslated regions).

[0103] Given the coding strand sequences encoding the stem cell growthfactor-like protein disclosed herein, antisense nucleic acids of theinvention can be designed according to the rules of Watson and Crick orHoogsteen base pairing. The antisense nucleic acid molecule can becomplementary to the entire coding region of stem cell growthfactor-like mRNA, but more preferably is an oligonucleotide that isantisense to only a portion of the coding or noncoding region of stemcell growth factor-like mRNA. For example, the antisense oligonucleotidecan be complementary to the region surrounding the translation startsite of stem cell growth factor-like mRNA. An antisense oligonucleotidecan be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50nucleotides in length. An antisense nucleic acid of the invention can beconstructed using chemical synthesis or enzymatic ligation reactionsusing procedures known in the art. For example, an antisense nucleicacid (e.g., an antisense oligonucleotide) can be chemically synthesizedusing naturally occurring nucleotides or variously modified nucleotidesdesigned to increase the biological stability of the molecules or toincrease the physical stability of the duplex formed between theantisense and sense nucleic acids (e.g., phosphorothioate derivativesand acridine substituted nucleotides can be used).

[0104] Examples of modified nucleotides that can be used to generate theantisense nucleic acid include: 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following section).

[0105] The antisense nucleic acid molecules of the invention aretypically administered to a subject or generated in situ such that theyhybridize with or bind to cellular mRNA and/or genomic DNA encoding astem cell growth factor-like protein to thereby inhibit expression ofthe protein (e.g., by inhibiting transcription and/or translation). Thehybridization can be by conventional nucleotide complementarity to forma stable duplex, or, for example, in the case of an antisense nucleicacid molecule that binds to DNA duplexes, through specific interactionsin the major groove of the double helix. An example of a route ofadministration of antisense nucleic acid molecules of the inventionincludes direct injection at a tissue site. Alternatively, antisensenucleic acid molecules can be modified to target selected cells and thenadministered systemically. For example, for systemic administration,antisense molecules can be modified such that they specifically bind toreceptors or antigens expressed on a selected cell surface (e.g., bylinking the antisense nucleic acid molecules to peptides or antibodiesthat bind to cell surface receptors or antigens). The antisense nucleicacid molecules can also be delivered to cells using the vectorsdescribed herein. To achieve sufficient nucleic acid molecules, vectorconstructs in which the antisense nucleic acid molecule is placed underthe control of a strong pol II or pol III promoter are preferred.

[0106] In yet another embodiment, the antisense nucleic acid molecule ofthe invention is an alpha-anomeric nucleic acid molecule. Analpha-anomeric nucleic acid molecule forms specific double-strandedhybrids with complementary RNA in which, contrary to the usualalpha-units. the strands run parallel to each other. See, e.g.,Gaultier, et al., 1987, Nucl. Acids Res. 15: 6625-6641. The antisensenucleic acid molecule can also comprise a 2′-o-methylribonucleotide(see, e.g., Inoue, et al, 1987, Nucl. Acids Res. 15: 6131-6148) or achimeric RNA-DNA analogue (see, e.g., Inoue, et al., 1987, FEBS Lett.215: 327-330.

[0107] 4.2.2 Ribozymes and PNA Moieties

[0108] Nucleic acid modifications include, by way of non-limitingexample modified bases, and nucleic acids whose sugar phosphatebackbones are modified or derivatized. These modifications are carriedout at least in part to enhance the chemical stability of the modifiednucleic acid, such that they can be used, for example, as antisensebinding nucleic acids in therapeutic applications in a subject.

[0109] In one embodiment, an antisense nucleic acid of the invention isa ribozyme. Ribozymes are catalytic RNA molecules with ribonucleaseactivity that are capable of cleaving a single-stranded nucleic acid,such as an mRNA, to which they have a complementary region. Thus,ribozymes (e.g., hammerhead ribozymes as described in Haselhoff andGerlach 1988, Nature 334: 585-591) can be used to catalytically cleavestem cell growth factor-like mRNA transcripts to thereby inhibittranslation of stem cell growth factor-like mRNA. A ribozyme havingspecificity for a stem cell growth factor-like-encoding nucleic acid canbe designed based upon the nucleotide sequence of a stem cell growthfactor-like cDNA disclosed herein. For example, a derivative of aTetrahymena L-19 IVS RNA can be constructed in which the nucleotidesequence of the active site is complementary to the nucleotide sequenceto be cleaved in a stem cell growth factor-like-encoding mRNA. See,e.g., U.S. Pat. No. 4,987,071 to Cech, et al, and U.S. Pat. No.5,116,742 to Cech, et al. Stem cell growth factor-like mRNA can also beused to select a catalytic RNA having a specific ribonuclease activityfrom a pool of RNA molecules. See, e.g., Bartel et al., (1993) Science261:1411-1418.

[0110] Alternatively, stem cell growth factor-like gene expression canbe inhibited by targeting nucleotide sequences complementary to theregulatory region of the stem cell growth factor-like nucleic acid(e.g., the stem cell growth factor-like promoter and/or enhancers) toform triple helical structures that prevent transcription of the stemcell growth factor-like gene in target cells. See, e.g., Helene, 1991,Anticancer Drug Des. 6: 569-84; Helene, et al, 1992, Ann. N.Y. Acad.Sci. 660: 27-36; Maher, 1992, Bioassays 14: 807-15.

[0111] In various embodiments, the stem cell growth factor-like nucleicacids can be modified at the base moiety, sugar moiety or phosphatebackbone to improve, e.g., the stability, hybridization, or solubilityof the molecule. For example, the deoxyribose phosphate backbone of thenucleic acids can be modified to generate peptide nucleic acids. See,e.g., Hyrup, et al., 1996, Bioorg Med Chem 4: 5-23. As used herein, theterms “peptide nucleic acids” or “PNAs” refer to nucleic acid mimics(e.g., DNA mimics) in which the deoxyribose phosphate backbone isreplaced by a pseudopeptide backbone and only the four naturalnucleobases are retained. The neutral backbone of PNAs has been shown toallow for specific hybridization to DNA and RNA under conditions of lowionic strength. The synthesis of PNA oligomers can be performed usingstandard solid phase peptide synthesis protocols as described in Hyrup,et al., 1996, supra; Perry-O'Keefe, et al., 1996, Proc. Natl. Acad. Sci.USA 93: 14670-14675.

[0112] PNAs of stem cell growth factor-like can be used in therapeuticand diagnostic applications. For example, PNAs can be used as antisenseor antigene agents for sequence-specific modulation of gene expressionby, e.g., inducing transcription or translation arrest or inhibitingreplication. PNAs of stem cell growth factor-like can also be used, forexample, in the analysis of single base pair mutations in a gene (e.g.,PNA directed PCR clamping; as artificial restriction enzymes when usedin combination with other enzymes, e.g., S1 nucleases (see, Hyrup, etal., 1996, supra); or as probes or primers for DNA sequence andhybridization (see, Hyrup, et al., 1996, supra; Perry-O'Keefe, et al.,1996, supra).

[0113] In another embodiment, PNAs of stem cell growth factor-like canbe modified, e g., to enhance their stability or cellular uptake, byattaching lipophilic or other helper groups to PNA, by the formation ofPNA-DNA chimeras, or by the use of liposomes or other techniques of drugdelivery known in the art. For example, PNA-DNA chimeras of stem cellgrowth factor-like can be generated that may combine the advantageousproperties of PNA and DNA. Such chimeras allow DNA recognition enzymes(e.g., RNase H and DNA polymerases) to interact with the DNA portionwhile the PNA portion would provide high binding affinity andspecificity. PNA-DNA chimeras can be linked using linkers of appropriatelengths selected in terms of base stacking, number of bonds between thenucleobases, and orientation (see. Hyrup, et al., 1996, supra). Thesynthesis of PNA-DNA chimeras can be performed as described in Hyrup, etal., 1996, Supra, et al., 1996, Nucl Acids Res 24: 3357-3363. Forexample, a DNA chain can be synthesized on a solid support usingstandard phosphoramidite coupling chemistry, and modified nucleosideanalogs, e.g., 5′-(4-methoxytrityl)amino-5′-deoxy-thymidinephosphoramidite, can be used between the PNA and the 5′ end of DNA. See,e.g., Mag, et al., 1989, Nucl Acid Res 17: 5973-5988. PNA monomers arethen coupled in a stepwise manner to produce a chimeric molecule with a5′ PNA segment and a 3′ DNA segment. See, e.g., Finn, et al., 1996,supra. Alternatively, chimeric molecules can be synthesized with a 5′DNA segment and a 3′ PNA segment. See, e.g., Petersen, et al., 1975,Bioorg. Med. Chem. Lett. 5: 1119-11124.

[0114] In other embodiments, the oligonucleotide may include otherappended groups such as peptides (e.g., for targeting host cellreceptors in vivo), or agents facilitating transport across the cellmembrane (see, e.g., Letsinger, et al., 1989, Proc. Natl. Acad. Sci.U.S.A. 86: 6553-6556; Lemaitre, et al., 1987, Proc. Natl. Acad. Sci. 84:648-652; PCT Publication No. WO88/09810) or the blood-brain barrier(see, e.g., PCT Publication No. WO 89/10134). In addition,oligonucleotides can be modified with hybridization-triggered cleavageagents (see, e.g., Krol, et al., 1988, BioTechniques 6:958-976) orintercalating agents (see, e.g., Zon, 1988, Pharm. Res. 5: 539-549). Tothis end, the oligonucleotide can be conjugated to another molecule,e.g., a peptide, a hybridization triggered cross-linking, agent, atransport agent, a hybridization-triggered cleavage agent, and the like.

4.3 Hosts

[0115] The present invention further provides host cells geneticallyengineered to contain the polynucleotides of the invention. For example,such host cells may contain nucleic acids of the invention introducedinto the host cell using known transformation, transfection or infectionmethods. The present invention still further provides host cellsgenetically engineered to express the polynucleotides of the invention,wherein such polynucleotides are in operative association with aregulatory sequence heterologous to the host cell which drivesexpression of the polynucleotides in the cell.

[0116] The host cell can be a higher eukaryotic host cell, such as amammalian cell, a lower eukaryotic host cell, such as a yeast cell, orthe host cell can be a prokaryotic cell, such as a bacterial cell.Introduction of the recombinant construct into the host cell can beeffected by calcium phosphate transfection, DEAE, dextran mediatedtransfection, or electroporation (Davis, L. et al., Basic Methods inMolecular Biology (1986)). The host cells containing one ofpolynucleotides of the invention, can be used in conventional manners toproduce the gene product encoded by the isolated fragment (in the caseof an ORF) or can be used to produce a heterologous protein under thecontrol of the EMF.

[0117] Any host/vector system can be used to express one or more of theORFs of the present invention. These include, but are not limited to,eukaryotic hosts such as HeLa cells, Cv-1 cell, COS cells, and Sf9cells, as well as prokaryotic host such as E. coli and B. subtilis. Themost preferred cells are those which do not normally express theparticular polypeptide or protein or which expresses the polypeptide orprotein at low natural level. Mature proteins can be expressed inmammalian cells, yeast, bacteria, or other cells under the control ofappropriate promoters. Cell-free translation systems can also beemployed to produce such proteins using RNAs derived from the DNAconstructs of the present invention. Appropriate cloning and expressionvectors for use with prokaryotic and eukaryotic hosts are described bySambrook, et al., in Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor, N.Y. (1989), the disclosure of which ishereby incorporated by reference.

[0118] Various mammalian cell culture systems can also be employed toexpress recombinant protein. Examples of mammalian expression systemsinclude the COS-7 lines of monkey kidney fibroblasts, described byGluzman, Cell 23:175 (1981), and other cell lines capable of expressinga compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK celltines. Mammalian expression vectors will comprise an origin ofreplication, a suitable promoter, and also any necessary ribosomebinding sites, polyadenylation site, splice donor and acceptor sites,transcriptional termination sequences, and 5′ flanking nontranscribedsequences. DNA sequences derived from the SV40 viral genome, forexample, SV40 origin, early promoter, enhancer, splice, andpolyadenylation sites may be used to provide the required nontranscribedgenetic elements. Recombinant polypeptides and proteins produced inbacterial culture are usually isolated by initial extraction from cellpellets, followed by one or more salting-out, aqueous ion exchange orsize exclusion chromatography steps. Protein refolding steps can beused, as necessary, in completing configuration of the mature protein.Finally, high performance liquid chromatography (HPLC) can be employedfor final purification steps. Microbial cells employed in expression ofproteins can be disrupted by any convenient method, includingfreeze-thaw cycling, sonication, mechanical disruption, or use of celllysing agents.

[0119] A number of types of cells may act as suitable host cells forexpression of the protein. Mammalian host cells include, for example,monkey COS cells, Chinese Hamster Ovary (CHO) cells, human kidney 293cells, human epidermal A431 cells, human Colo205 cells, 3T3 cells, CV-1cells, other transformed primate cell lines, normal diploid cells, cellstrains derived from in vitro culture of primary tissue, primaryexplants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK or Jurkatcells.

[0120] Alternatively, it may be possible to produce the protein in lowereukaryotes such as yeast or in prokaryotes such as bacteria. Potentiallysuitable yeast strains include Saccharomyces cerevisiae,Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeaststrain capable of expressing heterologous proteins. Potentially suitablebacterial strains include Escherichia coli, Bacillus subtilis,Salmonella typhimurium, or any bacterial strain capable of expressingheterologous proteins. If the protein is made in yeast or bacteria, itmay be necessary to modify the protein produced therein for example byphosphorylation or glycosylation of the appropriate sites, in order toobtain the functional protein. Such covalent attachments may beaccomplished using known chemical or enzymatic methods.

[0121] In another embodiment of the present invention, cells and tissuesmay be engineered to express an endogenous gene comprising thepolynucleotides of the invention under the control of inducibleregulatory elements, in which case the regulatory sequences of theendogenous gene may be replaced by homologous recombination. Asdescribed herein, gene targeting can be used to replace a gene'sexisting regulatory region with a regulatory sequence isolated from adifferent gene or a novel regulatory sequence synthesized by geneticengineering methods. Such regulatory sequences may be comprised ofpromoters, enhancers, scaffold-attachment regions, negative regulatorelements, transcriptional initiation sites, regulatory protein bindingsites or combinations of said sequences. Alternatively, sequences whichaffect the structure or stability of the RNA or protein produced may bereplaced, removed, added, or otherwise modified by targeting, includingpolyadenylation signals, mRNA stability elements, splice sites, leadersequences for enhancing or modifying transport or secretion propertiesof the protein, or other sequences which alter or improve the functionor stability of protein or RNA molecules.

[0122] The targeting event may be a simple insertion of the regulatorysequence, placing the gene under the control of the new regulatorysequence, e.g., inserting a new promoter or enhancer or both upstream ofa gene. Alternatively, the targeting event may be a simple deletion of aregulatory element, such as the deletion of a tissue-specific negativeregulatory element. Alternatively, the targeting event may replace anexisting element; for example, a tissue-specific enhancer can bereplaced by an enhancer that has broader or different cell-typespecificity than the naturally occurring elements. Here, the naturallyoccurring sequences are deleted and new sequences are added. In allcases, the identification of the targeting event may be facilitated bythe use of one or more selectable marker genes that are contiguous withthe targeting DNA, allowing for the selection of cells in which theexogenous DNA has integrated into the host cell genome. Theidentification of the targeting event may also be facilitated by the useof one or more marker genes exhibiting the property of negativeselection, such that the negatively selectable marker is linked to theexogenous DNA, but configured such that the negatively selectable markerflanks the targeting sequence, and such that a correct homologousrecombination event with sequences in the host cell genome does notresult in the stable integration of the negatively selectable marker.Markers useful for this purpose include the Herpes Simplex Virusthymidine kinase (TK) gene or the bacterial xanthine-guaninephosphoribosyl-transferase (gpt) gene.

[0123] The gene targeting or gene activation techniques which can beused in accordance with this aspect of the invention are moreparticularly described in U.S. Pat. No. 5,272,071 to Chappel; U.S. Pat.No. 5,578,461 to Sherwin et al.; International Application No.PCT/US92/09627 (WO93/09222) by Selden et al.; and InternationalApplication No. PCT/US90/06436 (WO91/06667) by Skoultchi et al., each ofwhich is incorporated by reference herein in its entirety.

[0124] 4.3.1 Chimeric and Fusion Proteins

[0125] The invention also provides stem cell growth factor-like chimericor fusion proteins. As used herein, a stem cell growth factor-like“chimeric protein” or “fusion protein” comprises a stem cell growthfactor-like polypeptide operatively-linked to a non-stem cell growthfactor-like polypeptide. A “stem cell growth factor-like polypeptide”refers to a polypeptide having an amino acid sequence corresponding to astem cell growth factor-like protein, whereas a “non-stem cell growthfactor-like polypeptide” refers to a polypeptide having an amino acidsequence corresponding to a protein that is not substantially homologousto the stem cell growth factor-like protein, e.g., a protein that isdifferent from the stem cell growth factor-like protein and that isderived from the same or a different organism. Within a stem cell growthfactor-like fusion protein the stem cell growth factor-like polypeptidecan correspond to all or a portion of a stem cell growth factor-likeprotein. In one embodiment, a stem cell growth factor-like fusionprotein comprises at least one biologically active portion of a stemcell growth factor-like protein. In another embodiment, a stem cellgrowth factor-like fusion protein comprises at least two biologicallyactive portions of a stem cell growth factor-like protein. In yetanother embodiment, a stem cell growth factor-like fusion proteincomprises at least three biologically active portions of a stem cellgrowth factor-like protein. Within the fusion protein, the term“operatively-linked” is intended to indicate that the stem cell growthfactor-like polypeptide and the non-stem cell growth factor-likepolypeptide are fused in-frame with one another. The non-stem cellgrowth factor-like polypeptide can be fused to the N-terminus orC-terminus of the stem cell growth factor-like polypeptide.

[0126] In one embodiment, the fusion protein is a GST-stem cell crouchfactor-like fusion protein in which the stem cell growth factor-likesequences are fused to the C-terminus of the GST (glutathioneS-transferase) sequences. Such fusion proteins can facilitate thepurification of recombinant stem cell growth factor-like polypeptides.In another embodiment, the fusion protein is a stem cell growthfactor-like protein containing a heterologous signal sequence at itsN-terminus. In certain host cells (e.g., mammalian host cells),expression and/or secretion of stem cell growth factor-like can beincreased through use of a heterologous signal sequence.

[0127] In yet another embodiment, the fusion protein is a stem cellgrowth factor-like-immunoglobulin fusion protein in which the stem cellgrowth factor-like sequences are fused to sequences derived from amember of the immunoglobulin protein family. The stem cell growthfactor-like-immunoglobulin fusion proteins of the invention can beincorporated into pharmaceutical compositions and administered to asubject to inhibit an interaction between a stem cell growth factor-likeligand and a stem cell growth factor-like protein on the surface of acell, to thereby suppress stem cell growth factor-like-mediated signaltransduction in vivo. The stem cell growth factor-like-immunoglobulinfusion proteins can be used to affect the bioavailability of a stem cellgrowth factor-like cognate ligand. Inhibition of the stem cell growthfactor-like ligand/stem cell growth factor-like interaction can beuseful therapeutically for both the treatment of proliferative anddifferentiative disorders, as well as modulating (e.g. promoting orinhibiting) cell survival. Moreover, the stem cell growthfactor-like-immunoglobulin fusion proteins of the invention can be usedas immunogens to produce anti-stem cell growth factor-like antibodies ina subject, to purify stem cell growth factor-like ligands, and inscreening assays to identify molecules that inhibit the interaction ofstem cell growth factor-like with a stem cell growth factor-like ligand.

[0128] A stem cell growth factor-like chimeric or fusion protein of theinvention can be produced by standard recombinant DNA techniques. Forexample, DNA fragments coding for the different polypeptide sequencesare ligated together in-frame in accordance with conventionaltechniques, e.g., by employing blunt-ended or stagger-ended termini forligation, restriction enzyme digestion to provide for appropriatetermini, filling-in of cohesive ends as appropriate, alkalinephosphatase treatment to avoid undesirable joining, and enzymaticligation. In another embodiment, the fusion gene can be synthesized byconventional techniques including automated DNA synthesizers.Alternatively, PCR amplification of gene fragments can be carried outusing anchor primers that give rise to complementary overhangs betweentwo consecutive gene fragments that can subsequently be annealed andreamplified to generate a chimeric gene sequence (see, e.g., Ausubel, etal, (eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons,1992). Moreover, many expression vectors are commercially available thatalready encode a fusion moiety (e.g., a GST polypeptide). A stem cellgrowth factor-like-encoding nucleic acid can be cloned into such anexpression vector such that the fusion moiety is linked in-frame to thestem cell growth factor-like protein.

4.4 Polypeptides of the Invention

[0129] The isolated polypeptides of the invention include, but are notlimited to, a polypeptide comprising: the amino acid sequence set forthas any one of SEQ ID NO: 23, 25, 28, 30-32, 34, or 35 or an amino acidsequence encoded by any one of the nucleotide sequences SEQ ID NO: 1-22,24, 26-27, 29, or 33 or the corresponding full length or mature protein.Polypeptides of the invention also include polypeptides preferably withbiological or immunological activity that are encoded by: (a) apolynucleotide having any one of the nucleotide sequences set forth inSEQ ID NO: 1-24, 26-27, 29, or 33 or (b) polynucleotides encoding anyone of the amino acid sequences set forth as SEQ ID NO: 23, 25, 28,30-32, 34, or 35 or (c) polynucleotides that hybridize to the complementof the polynucleotides of either (a) or (b) under stringenthybridization conditions. The invention also provides biologicallyactive or immunologically active variants of any of the amino acidsequences set forth as SEQ ID NO: 23, 25, 28, 30-32, 34 or 35 or thecorresponding full length or mature protein; and “substantialequivalents” thereof (e.g., with at least about 65%, at least about 70%,at least about 75%, at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, or 89%, more typically at least about 90%, 91%, 92%, 93%, or94% and even more typically at least about 95%, 96%, 97%, 98% or 99%,most typically at least about 99% amino acid identity) that retainbiological activity. Polypeptides encoded by allelic variants may have asimilar, increased, or decreased activity compared to polypeptidescomprising SEQ ID NO: 23, 25, 28, 30-32, 34, or 35.

[0130] Fragments of the proteins of the present invention which arecapable of exhibiting biological activity are also encompassed by thepresent invention. Fragments of the protein may be in linear form orthey may be cyclized using known methods, for example, as described inH. U. Saragovi, et al., Bio/Technology 10, 773-778 (1992) and in R. S.McDowell, et al., J. Amer. Chem. Soc. 114, 9245-9253 (1992), both ofwhich are incorporated herein by reference. Such fragments may be fusedto carrier molecules such as immunoglobulins for many purposes,including increasing the valency of protein binding sites.

[0131] The present invention also provides both full-length and matureforms (for example, without a signal sequence or precursor sequence) ofthe disclosed proteins. The protein coding sequence is identified in thesequence listing by translation of the disclosed nucleotide sequences.The mature form of such protein may be obtained by expression of afull-length polynucleotide in a suitable mammalian cell or other hostcell. The sequence of the mature form of the protein is alsodeterminable from the amino acid sequence of the full-length form. Whereproteins of the present invention are membrane bound, soluble forms ofthe proteins are also provided. In such forms, part or all of theregions causing the proteins to be membrane bound are deleted so thatthe proteins are fully secreted from the cell in which it is expressed.

[0132] Protein compositions of the present invention may furthercomprise an acceptable carrier, such as a hydrophilic, e.g.,pharmaceutically acceptable, carrier.

[0133] The present invention further provides isolated polypeptidesencoded by the nucleic acid fragments of the present invention or bydegenerate variants of the nucleic acid fragments of the presentinvention. By “degenerate variant” is intended nucleotide fragmentswhich differ from a nucleic acid fragment of the present invention(e.g., an ORF) by nucleotide sequence but, due to the degeneracy of thegenetic code, encode an identical polypeptide sequence. Preferrednucleic acid fragments of the present invention are the ORFs that encodeproteins.

[0134] A variety of methodologies known in the art can be utilized toobtain any one of the isolated polypeptides or proteins of the presentinvention. At the simplest level, the amino acid sequence can besynthesized using commercially available peptide synthesizers. Thesynthetically-constructed protein sequences, by virtue of sharingprimary, secondary or tertiary structural and/or conformationalcharacteristics with proteins may possess biological properties incommon therewith, including protein activity. This technique isparticularly useful in producing small peptides and fragments of largerpolypeptides. Fragments are useful, for example, in generatingantibodies against the native polypeptide. Thus, they may be employed asbiologically active or immunological substitutes for natural, purifiedproteins in screening of therapeutic compounds and in immunologicalprocesses for the development of antibodies.

[0135] The polypeptides and proteins of the present invention canalternatively be purified from cells which have been altered to expressthe desired polypeptide or protein. As used herein, a cell is said to bealtered to express a desired polypeptide or protein when the cell,through genetic manipulation, is made to produce a polypeptide orprotein which it normally does not produce or which the cell normallyproduces at a lower level. One skilled in the art can readily adaptprocedures for introducing and expressing either recombinant orsynthetic sequences into eukaryotic or prokaryotic cells in order togenerate a cell which produces one of the polypeptides or proteins ofthe present invention.

[0136] The invention also relates to methods for producing a polypeptidecomprising growing a culture of host cells of the invention in asuitable culture medium, and purifying the protein from the cells or theculture in which the cells are grown. For example, the methods of theinvention include a process for producing a polypeptide in which a hostcell containing a suitable expression vector that includes apolynucleotide of the invention is cultured under conditions that allowexpression of the encoded polypeptide. The polypeptide can be recoveredfrom the culture, conveniently from the culture medium, or from a lysateprepared from the host cells and further purified. Preferred embodimentsinclude those in which the protein produced by such process is a fulllength or mature form of the protein.

[0137] In an alternative method, the polypeptide or protein is purifiedfrom bacterial cells which naturally produce the polypeptide or protein.One skilled in the art can readily follow known methods for isolatingpolypeptides and proteins in order to obtain one of the isolatedpolypeptides or proteins of the present invention. These include, butare not limited to, immunochromatography, HPLC, size-exclusionchromatography, ion-exchange chromatography, and immuno-affinitychromatography. See, e.g., Scopes. Protein Purification: Principles andPractice, Springer-Verlag (1994); Sambrook, et al., in MolecularCloning: A Laboratory Manual; Ausubel et al., Current Protocols inMolecular Biology. Polypeptide fragments that retainbiological/immunological activity include fragments comprising greaterthan about 100 amino acids, or greater than about 200 amino acids, andfragments that encode specific protein domains.

[0138] The purified polypeptides can be used in in vitro binding assayswhich are well known in the art to identify molecules which bind to thepolypeptides. These molecules include but are not limited to, for e.g.,small molecules, molecules from combinatorial libraries, antibodies orother proteins. The molecules identified in the binding assay are thentested for antagonist or agonist activity in in vivo tissue culture oranimal models that are well known in the art. In brief, the moleculesare titrated into a plurality of cell cultures or animals and thentested for either cell/animal death or prolonged survival of theanimal/cells.

[0139] In addition, the peptides of the invention or molecules capableof binding to the peptides may be complexed with toxins, e.g., ricin orcholera, or with other compounds that are toxic to cells. Thetoxin-binding molecule complex is then targeted to a tumor or other cellby the specificity of the binding molecule for SEQ ID NO: 23, 25, 28,30-32, 34, or 35.

[0140] The protein of the invention may also be expressed as a productof transgenic animals, e.g., as a component of the milk of transgeniccows, goats, pigs, or sheep which are characterized by somatic or germcells containing a nucleotide sequence encoding the protein.

[0141] The proteins provided herein also include proteins characterizedby amino acid sequences similar to those of purified proteins but intowhich modification are naturally provided or deliberately engineered.For example, modifications, in the peptide or DNA sequence, can be madeby those skilled in the art using known techniques. Modifications ofinterest in the protein sequences may include the alteration,substitution, replacement, insertion or deletion of a selected aminoacid residue in the coding sequence. For example, one or more of thecysteine residues may be deleted or replaced with another amino acid toalter the conformation of the molecule. Techniques for such alteration,substitution, replacement, insertion or deletion are well known to thoseskilled in the art (see, e.g., U.S. Pat. No. 4,518,584). Preferably,such alteration, substitution, replacement, insertion or deletionretains the desired activity of the protein. Regions of the protein thatare important for the protein function can be determined by variousmethods known in the art including the alanine-scanning method whichinvolved systematic substitution of single or strings of amino acidswith alanine, followed by testing the resulting alanine-containingvariant for biological activity. This type of analysis determines theimportance of the substituted amino acid(s) in biological activity.Regions of the protein that are important for protein function may bedetermined by the eMATRIX program.

[0142] Other fragments and derivatives of the sequences of proteinswhich would be expected to retain protein activity in whole or in partand are useful for screening or other immunological methodologies mayalso be easily made by those skilled in the art given the disclosuresherein. Such modifications are encompassed by the present invention.

[0143] The protein may also be produced by operably linking the isolatedpolynucleotide of the invention to suitable control sequences in one ormore insect expression vectors, and employing an insect expressionsystem. Materials and methods for baculovirus/insect cell expressionsystems are commercially available in kit form from, e.g. Invitrogen,San Diego. Calif., U.S.A. (the MaxBat™ kit), and such methods are wellknown in the art, as described in Summers and Smith, Texas AgriculturalExperiment Station Bulletin No. 1555 (1987), incorporated herein byreference. As used herein, an insect cell capable of expressing apolynucleotide of the present invention is “transformed.”

[0144] The protein of the invention may be prepared by culturingtransformed host cells under culture conditions suitable to express therecombinant protein. The resulting expressed protein may then bepurified from such culture (i.e., from culture medium or cell extracts)using known purification processes, such as gel filtration and ionexchange chromatography. The purification of the protein may alsoinclude an affinity column containing agents which will bind to theprotein; one or more column steps over such affinity resins asconcanavalin A-agarose, heparin-toyopearl™ or Cibacrom blue 3GASepharose™; one or more steps involving hydrophobic interactionchromatography using such resins as phenyl ether, butyl ether, or propylether; or immunoaffinity chromatography.

[0145] Alternatively, the protein of the invention may also be expressedin a form which will facilitate purification. For example, it may beexpressed as a fusion protein, such as those of maltose binding protein(MBP), glutathione-S-transferase (GST) or thioredoxin (TRX), or as a Histag. Kits for expression and purification of such fusion proteins arecommercially available from New England BioLab (Beverly, Mass.),Pharmacia (Piscataway, N.J.) and Invitrogen, respectively. The proteincan also be tagged with an epitope and subsequently purified by using aspecific antibody directed to such epitope. One such epitope (“FLAGS®”)is commercially available from Kodak (New Haven, Conn.).

[0146] Finally, one or more reverse-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,e.g., silica gel having pendant methyl or other aliphatic groups, can beemployed to further purify the protein. Some or all of the foregoingpurification steps, in various combinations, can also be employed toprovide a substantially homogeneous isolated recombinant protein. Theprotein thus purified is substantially free of other mammalian proteinsand is defined in accordance with the present invention as an “isolatedprotein.”

[0147] The polypeptides of the invention include analogs (variants). Thepolypeptides of the invention include stem cell growth factor-likeanalogs. This embraces fragments of stem cell growth factor-likepolypeptide of the invention, as well stem cell growth factor-likepolypeptides which comprise one or more amino acids deleted, inserted,or substituted. Also, analogs of the stem cell growth factor-likepolypeptide of the invention embrace fusions of the stem cell growthfactor-like polypeptides or modifications of the stem cell growthfactor-like polypeptides, wherein the stem cell growth factor-likepolypeptide or analog is fused to another moiety or moieties, e.g.,targeting moiety or another therapeutic agent. Such analogs may exhibitimproved properties such as activity and/or stability. Examples ofmoieties which may be fused to the stem cell growth factor-likepolypeptide or an analog include, for example, targeting moieties whichprovide for the delivery of polypeptide to neurons, e.g., antibodies tocentral nervous system, or antibodies to receptor and ligands expressedon neuronal cells. Other moieties which may be fused to stem cell growthfactor-like polypeptide include therapeutic agents which are used fortreatment, for example anti-depressant drugs or other medications forneurological disorders. Also, stem cell growth factor-like polypeptidesmay be fused to neuron growth modulators, and other chemokines fortargeted delivery.

[0148] 4.4.1 Determining Polypeptide and Polynucleotide Identity andSimilarity

[0149] Preferred identity and/or similarity are designed to give thelargest match between the sequences tested. Methods to determineidentity and similarity are codified in computer programs including, butare not limited to, the GCG program package, including GAP (Devereux,J., et al., Nucleic Acids Research 12(1):387 (1984); Genetics ComputerGroup, University of Wisconsin, Madison, Wis.). BLASTP, BLASTN, BLASTX,FASTA (Altschul, S. F. et al., J. Molec. Biol. 215:403-410 (1990).PSI-BLAST (Altschul S. F. et al., Nucleic Acids Res. vol. 25, pp3389-3402, herein incorporated by reference), the eMatrix software (Wuet al., J. Comp. Biol., vol. 6, pp 219-235 (1999), herein incorporatedby reference), eMotif software (Nevill-Manninc et al, ISMB-97, vol 4, pp202-209, herein incorporated by reference), the GeneAtlas software(Molecular Simulations Inc. (MSI), San Diego, Calif.) (Sanchez and Sali(1998) Proc. Natl. Acad. Sci., 95, 13597-13602; Kitson D H et al, (2000)“Remote homology detection using structural modeline—an evaluation”Submitted; Fischer and Eisenberg (1996) Protein Sci. 5, 947-955), andthe Kyte-Doolittle hydrophobocity prediction algorithm (J. Mol Biol.157, pp. 105-31 (1982), incorporated herein by reference). The BLASTprograms are publicly available from the National Center forBiotechnology Information (NCBI) and other sources (BLAST Manual,Altschul, S., et al, NCB NLM NIH Bethesda, Md. 20894; Altschul. S., etal., J. Mol. Biol. 215:403-410 (1990).

4.5 Gene Therapy

[0150] Mutations in the polynucleotides of the invention gene may resultin loss of normal function of the encoded protein. The invention thusprovides gene therapy to restore normal activity of the polypeptides ofthe invention; or to treat disease states involving polypeptides of theinvention. Delivery of a functional gene encodin polypeptides of theinvention to appropriate cells is effected ex vivo, in situ, or in vivoby use of vectors, and more particularly viral vectors (e.g.,adenovirus, adeno-associated virus, or a retrovirus), or ex vivo by useof physical DNA transfer methods (e.g., liposomes or chemicaltreatments). See, for example, Anderson, Nature, supplement to vol. 392,no. 6679, pp.25-20 (1998). For additional reviews of gene therapytechnology see Friedmann, Science, 244: 1275-1281 (1989); Verma,Scientific American: 68-84 (1990); and Miller, Nature. 357: 455-460(1992). Introduction of any one of the nucleotides of the presentinvention or a gene encoding the polypeptides of the present inventioncan also be accomplished with extrachromosomal substrates (transientexpression) or artificial chromosomes (stable expression). Cells mayalso be cultured ex vivo in the presence of proteins of the presentinvention in order to proliferate or to produce a desired effect on oractivity in such cells. Treated cells can then be introduced in vivo fortherapeutic purposes. Alternatively, it is contemplated that in otherhuman disease states, preventing the expression of or inhibiting theactivity of polypeptides of the invention will be useful in treating thedisease states. It is contemplated that antisense therapy or genetherapy could be applied to negatively regulate the expression ofpolypeptides of the invention.

[0151] Other methods inhibiting expression of a protein include theintroduction of antisense molecules to the nucleic acids of the presentinvention, their complements, or their translated RNA sequences, bymethods known in the art. Further, the polypeptides of the presentinvention can be inhibited by using targeted deletion methods, or theinsertion of a negative regulatory element such as a silencer, which istissue specific.

[0152] The present invention still further provides cells geneticallyengineered in vivo to express the polynucleotides of the invention,wherein such polynucleotides are in operative association with aregulator, sequence heterologous to the host cell which drivesexpression of the polynucleotides in the cell. These methods can be usedto increase or decrease the expression of the polynucleotides of thepresent invention.

[0153] Knowledge of DNA sequences provided by the invention allows formodification of cells to permit, increase, or decrease, expression ofendogenous polypeptide. Cells can be modified (e.g., by homologousrecombination) to provide increased polypeptide expression by replacing,in whole or in part, the naturally occurring promoter with all or partof a heterologous promoter so that the cells express the protein athigher levels. The heterologous promoter is inserted in such a mannerthat it is operatively linked to the desired protein encoding sequences.See, for example, PCT International Publication No. WO 94/12650, PCTInternational Publication No. WO 92/20808, and PCT InternationalPublication No. WO 91/09955. It is also contemplated that, in additionto heterologous promoter DNA, amplifiable marker DNA (e.g., ada, dhfr,and the multifunctional CAD gene which encodes carbamyl phosphatesynthase, aspartate transcarbamylase, and dihydroorotase) and/or intronDNA may be inserted along with the heterologous promoter DNA. If linkedto the desired protein coding sequence, amplification of the marker DNAby standard selection methods results in co-amplification of the desiredprotein coding sequences in the cells.

[0154] In another embodiment of the present invention, cells and tissuesmay be engineered to express an endogenous gene comprising thepolynucleotides of the invention under the control of inducibleregulatory elements, in which case the regulatory sequences of theendogenous gene may be replaced by homologous recombination. Asdescribed herein, gene targeting can be used to replace a gene'sexisting regulatory region with a regulatory sequence isolated from adifferent gene or a novel regulatory sequence synthesized by geneticengineering methods. Such regulatory sequences may be comprised ofpromoters, enhancers, scaffold-attachment regions, negative regulatoryelements, transcriptional initiation sites, regulatory protein bindingsites or combinations of said sequences. Alternatively, sequences whichaffect the structure or stability of the RNA or protein produced may bereplaced, removed, added, or otherwise modified by targeting. Thesesequences include polyadenylation signals, mRNA stability elements,splice sites, leader sequences for enhancing or modifying transport orsecretion properties of the protein, or other sequences which alter orimprove the function or stability of protein or RNA molecules.

[0155] The targeting event may be a simple insertion of the regulatorysequence, placing the gene under the control of the new regulator,sequence, e.g., inserting a new promoter or enhancer or both upstream ofa gene. Alternatively, the targeting event may be a simple deletion of aregulatory element, such as the deletion of a tissue-specific negativerepulatory element. Alternatively, the targeting event may replace anexisting element; for example, a tissue-specific enhancer can bereplaced by an enhancer that has broader or different cell-typespecificity than the naturally occurring elements. Here, the naturallyoccurring sequences are deleted and nest sequences are added. In allcases, the identification of the targeting event may be facilitated bythe use of one or more selectable marker genes that are contiguous withthe targeting DNA, allowing for the selection of cells in which theexogenous DNA has integrated into the cell genome. The identification ofthe targeting event may also be facilitated by the use of one or moremarker genes exhibiting the property of negative selection, such thatthe negatively selectable marker is linked to the exogenous DNA, butconfigured such that the negatively selectable marker flanks thetargeting sequence, and such that a correct homologous recombinationevent with sequences in the host cell genome does not result in thestable integration of the negatively selectable marker. Markers usefulfor this purpose include the Herpes Simplex Virus thymidine kinase (TK)gene or the bacterial xanthine-guanine phosphoribosyl-transferase(gpt)gene.

[0156] The gene targeting or gene activation techniques which can beused in accordance with this aspect of the invention are moreparticularly described in U.S. Pat. No. 5,272,071 to Chappel; U.S. Pat.No. 5,578,461 to Sherwin et al.; International Application No.PCT/US92/09627 (WO93/09222) by Selden et al.; and InternationalApplication No. PCT/US90/06436 (WO91/06667) by Skoultchi et al., each ofwhich is incorporated by reference herein in its entirety.

4.6 Transgenic Animals

[0157] In preferred methods to determine biological functions of thepolypeptides of the invention in vivo, one or more genes provided by theinvention are either over expressed or inactivated in the germ line ofanimals using homologous recombination [Capecchi, Science 244:1288-1292(1989)]. Animals in which the gene is over expressed, under theregulatory control of exogenous or endogenous promoter elements, areknown as transgenic animals. Animals in which an endogenous gene hasbeen inactivated by homologous recombination are referred to as“knockout” animals. Knockout animals, preferably non-human mammals, canbe prepared as described in U.S. Pat. No. 5,557,032, incorporated hereinby reference. Transgenic animals are useful to determine the rolespolypeptides of the invention play in biological processes, andpreferably in disease states. Transgenic animals are useful as modelsystems to identify compounds that modulate lipid metabolism. Transgenicanimals, preferably non-human mammals, are produced using methods asdescribed in U.S. Pat. No. 5,489,743 and PCT Publication No. WO94/28122,incorporated herein by reference.

[0158] Transgenic animals can be prepared wherein all or part of apromoter of the polynucleotides of the invention is either activated orinactivated to alter the level of expression of the polypeptides of theinvention. Inactivation can be carried out using homologousrecombination methods described above. Activation can be achieved bysupplementing or even replacing the homologous promoter to provide forincreased protein expression. The homologous promoter can besupplemented by insertion of one or more heterologous enhancer elementsknown to confer promoter activation in a particular tissue.

[0159] The polynucleotides of the present invention also make possiblethe development, through, e.g., homologous recombination or knock outstrategies; of animals that fail to express functional stem cell growthfactor-like polypeptide or that express a variant of stem cell growthfactor-like polypeptide. Such animals are useful as models for studyingthe in vivo activities of stem cell growth factor-like polypeptide aswell as for studying modulators of the stem cell growth factor-likepolypeptide.

[0160] In preferred methods to determine biological functions of thepolypeptides of the invention in vivo, one or more genes provided by theinvention are either over expressed or inactivated in the germ line ofanimals using homologous recombination [Capecchi, Science 244:1288-1292(1989)]. Animals in which the gene is over expressed, under theregulatory control of exogenous or endogenous promoter elements, areknown as transgenic animals. Animals in which an endogenous gene hasbeen inactivated by homologous recombination are referred to as“knockout” animals. Knockout animals, preferably non-human mammals, canbe prepared as described in U.S. Pat. No. 5,557,032, incorporated hereinby reference. Transgenic animals are useful to determine the rolespolypeptides of the invention play in biological processes, andpreferably in disease states. Transgenic animals are useful as modelsystems to identify compounds that modulate lipid metabolism. Transgenicanimals, preferably non-human mammals, are produced using methods asdescribed in U.S. Pat. No. 5,489,743 and PCT Publication No. WO94/28122,incorporated herein by reference.

[0161] Transgenic animals can be prepared wherein all or part of thepolynucleotides of the invention promoter is either activated orinactivated to alter the level of expression of the polypeptides of theinvention. Inactivation can be carried out using homologousrecombination methods described above. Activation can be achieved bysupplementing or even replacing the homologous promoter to provide forincreased protein expression. The homologous promoter can besupplemented by insertion of one or more heterologous enhancer elementsknown to confer promoter activation in a particular tissue.

4.7 Uses and Biological Activity of Human Stem Cell Growth Factor-LikePolypeptide

[0162] The polynucleotides and proteins of the present invention areexpected to exhibit one or more of the uses or biological activities(including those associated with assays cited herein) identified herein.Uses or activities described for proteins of the present invention maybe provided by administration or use of such proteins or ofpolynucleotides encoding such proteins (such as, for example, in genetherapies or vectors suitable for introduction of DNA). The mechanismunderlying the particular condition or pathology will dictate whetherthe polypeptides of the invention, the polynucleotides of the inventionor modulators (activators or inhibitors) thereof would be beneficial tothe subject in need of treatment. Thus, “therapeutic compositions of theinvention” include compositions comprising isolated polynucleotides(including recombinant DNA molecules, cloned genes and degeneratevariants thereof) or polypeptides of the invention (including fulllength protein, mature protein and truncations or domains thereof), orcompounds and other substances that modulate the overall activity of thetarget gene products, either at the level of target gene/proteinexpression or target protein activity. Such modulators includepolypeptides, analogs, (variants), including fragments and fusionproteins, antibodies and other binding proteins; chemical compounds thatdirectly or indirectly activate or inhibit the polypeptides of theinvention (identified, e.g., via drug screening assays as describedherein); antisense polynucleotides and polynucleotides suitable fortriple helix formation; and in particular antibodies or other bindingpartners that specifically recognize one or more epitopes of thepolypeptides of the invention.

[0163] The polypeptides of the present invention may likewise beinvolved in cellular activation or in one of the other physiologicalpathways described herein.

[0164] 4.7.1 Research Uses and Utilities

[0165] The polynucleotides provided by the present invention can be usedby the research community for various purposes. The polynucleotides canbe used to express recombinant protein for analysis, characterization ortherapeutic use; as markers for tissues in which the correspondingprotein is preferentially expressed (either constitutively or at aparticular stage of tissue differentiation or development or in diseasestates); as molecular weight markers on gels; as chromosome markers ortags (when labeled) to identify chromosomes or to map related genepositions; to compare with endogenous DNA sequences in patients toidentify potential genetic disorders; as probes to hybridize and thusdiscover novel, related DNA sequences; as a source of information toderive PCR primers for genetic fingerprinting; as a probe to“subtract-out” known sequences in the process of discovering other novelpolynucleotides; for selecting and making oligomers for attachment to a“gene chip” or other support, including for examination of expressionpatterns; to raise anti-protein antibodies using DNA immunizationtechniques; and as an antigen to raise anti-DNA antibodies or elicitanother immune response. Where the polynucleotide encodes a proteinwhich binds or potentially binds to another protein (such as, forexample, in a receptor-ligand interaction), the polynucleotide can alsobe used in interaction trap assays (such as, for example, that describedin Gyuris et al., Cell 75:791-803 (1993)) to identify polynucleotidesencoding the other protein with which binding occurs or to identifyinhibitors of the binding interaction.

[0166] The polypeptides provided by the present invention can similarlybe used in assays to determine biological activity, including in a panelof multiple proteins for high-throughput screening; to raise antibodiesor to elicit another immune response; as a reagent (including thelabeled reagent) in assays designed to quantitatively determine levelsof the protein (or its receptor) in biological fluids; as markers fortissues in which the corresponding polypeptide is preferentiallyexpressed (either constitutively or at a particular stage of tissuedifferentiation or development or in a disease state); and, of course,to isolate correlative receptors or ligands. Proteins involved in thesebinding interactions can also be used to screen for peptide or smallmolecule inhibitors or agonists of the binding interaction.

[0167] The polypeptides of the invention are also useful for makingantibody substances that are specifically immunoreactive with stem cellgrowth factor-like proteins. Antibodies and portions thereof (e.g., Fabfragments) which bind to the polypeptides of the invention can be usedto identify the presence of such polypeptides in a sample. Suchdeterminations are carried out using any suitable immunoassay format,and any polypeptide of the invention that is specifically bound by theantibody car be employed as a positive control.

[0168] Any or all of these research utilities are capable of beingdeveloped into reagent grade or kit format for commercialization asresearch products.

[0169] Methods for performing the uses listed above are well known tothose skilled in the art. References disclosing such methods includewithout limitation “Molecular Cloning: A Laboratory Manual”, 2d ed.,Cold Spring Harbor Laboratory Press, Sambrook. J., E. F. Fritsch and T.Maniatis eds., 1989, and “Methods in Enzymology: Guide to MolecularCloning Techniques”. Academic Press, Berger, S. L. and A. R. Kimmeleds., 1987.

[0170] 4.7.2 Nutritional Uses

[0171] Polynucleotides and polypeptides of the present invention canalso be used as nutritional sources or supplements. Such uses includewithout limitation use as a protein or amino acid supplement, use as acarbon source, use as a nitrogen source and use as a source ofcarbohydrate. In such cases the polypeptide or polynucleotide of theinvention can be added to the feed of a particular organism or can beadministered as a separate solid or liquid preparation, such as in theform of powder, pills, solutions, suspensions or capsules. In the caseof microorganisms, the polypeptide or polynucleotide of the inventioncan be added to the medium in or on which the microorganism is cultured.

[0172] Additionally, the polypeptides of the invention can be used asmarkers, and as a food supplement. A polypeptide consisting of SEQ IDNO: 34, for example, has a molecular mass of approximately 50.2 kDa inits unprocessed and unglycosylated state. Protein food supplements arewell known and the formulation of suitable food supplements includingpolypeptides of the invention is within the level of skill in the foodpreparation art.

[0173] 4.7.3 Cytokine and Cell Proliferation/Differentiation Activity

[0174] A polypeptide of the present invention may exhibit activityrelating to cytokine, cell proliferation (either inducing or inhibiting)or cell differentiation (either inducing or inhibiting) activity or mayinduce production of other cytokines in certain cell populations. Apolynucleotide of the invention can encode a polypeptide exhibiting suchattributes. Many protein factors discovered to date, including all knowncytokines, have exhibited activity in one or more factor-dependent cellproliferation assays, and hence the assays serve as a convenientconfirmation of cytokine activity. The activity of therapeuticcompositions of the present invention is evidenced by any one of anumber of routine factor dependent cell proliferation assays for celllines including, without limitation, 32D, DA2, DA1G, T10, B9, B9/11,BaF3, MC9/G, M+(preB M+), 2E8, RB5, DA1, 123, T1165, HT2, CTLL2, TF-1,Mo7e, CMK, HUVEC, and Caco. Therapeutic compositions of the inventioncan be used in the following:

[0175] Assays for T-cell or thymocyte proliferation include withoutlimitation those described in: Current Protocols in Immunology. Ed by J.E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober,Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, InVitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7.Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500,1986; Bertagnolli et al., J. Immunol. 145:1706-1712, 1990; Bertagnolliet al., Cellular Immunology 133:327-341, 1991; Bertagnolli, et al., I.Immunol. 149:3778-3783, 1992; Bowman et al., I. Immunol. 152:1756-1761,1994.

[0176] Assays for cytokine production and/or proliferation of spleencells, lymph node cells or thymocytes include, without limitation, thosedescribed in: Polyclonal T cell stimulation. Kruisbeek. A. M. andShevach. E. M. In Current Protocols in Immunology. J. E. e.a. Coliganeds. Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto, 1994; andMeasurement of mouse and human interleukin-γ. Schreiber, R. D. InCurrent Protocols in Immunology. J. E. e.a. Coligan eds. Vol 1 pp.6.8.1-6.8.8, John Wiley and Sons. Toronto, 1994.

[0177] Assays for proliferation and differentiation of hematopoietic andlymphopoietic cells include, without limitation, those described in:Measurement of Human and Murine Interleukin 2 and Interleukin 4.Bottomly. K., Davis, L. S. and Lipsky, P. E. In Current Protocols inImmunology. J. E. e.a. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John Wileyand Sons, Toronto, 1991; deVries et al., J. Exp. Med. 173:1205-1211,1991; Moreau et al., Nature 336:690-692, 1988; Greenberger et al., Proc.Natl. Acad. Sci. U.S.A. 80:2931-2938, 1983; Measurement of mouse andhuman interleukin 6—Nordan, R. In Current Protocols in Immunology. J. E.Coligan eds. Vol 1 pp. 6.6.1-6.6.5, John Wiley and Sons, Toronto, 1991;Smith et al., Proc. Natl. Aced. Sci. U.S.A. 83: 1857-1861, 1986;Measurement of human Interleukin II—Bennett. F., Giannotti, J., Clark,S. C. and Turner, K. J. In Current Protocols in Immunology. J. E.Coligan eds. Vol 1 pp. 6.15.1 John Wiley and Sons, Toronto, 1991;Measurement of mouse and human Interleukin 9-Ciarletta, A., Giannotti,J., Clark, S. C. and Turner, K. J. In Current Protocols in Immunology.J. E. Coligan eds. Vol 1 pp. 6.13.1, John Wiley and Sons, Toronto, 1991.

[0178] Assays for T-cell clone responses to antigens (which willidentify, among others, proteins that affect APC-T cell interactions aswell as direct T-cell effects by measuring proliferation and cytokineproduction) include, without limitation, those described in: CurrentProtocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H.Margulies, E. M. Shevach, W Strober, Pub. Greene Publishing Associatesand Wiley-Interscience (Chapter 3, In Vitro assays for Mouse LymphocyteFunction; Chapter 6, Cytokines and their cellular receptors; Chapter 7.Immunologic studies in Humans), Weinberger et al., Proc. Natl. Acad.Sci. USA 77:6091-6095, 1980; Weinberger et al., Eur. J. Immun.11:405-411, 1981; Takai et al., J. Immunol. 137:3494-3500, 1986; Takaiet al., J. Immunol. 140:508-512, 1988.

[0179] 4.7.4 Stem Cell Growth Factor Activity

[0180] A polypeptide of the present invention may exhibit stem cellgrowth factor activity and be involved in the proliferation,differentiation and survival of pluripotent and totipotent stem cellsincluding primordial germ cells, embryonic stem cells, hematopoieticstem cells and/or germ line stem cells. Administration of thepolypeptide of the invention to stem cells in vivo or ex vivo maymaintain and expand cell populations in a totipotential orpluripotential state which would be useful for re-engineering damaged ordiseased tissues, transplantation, manufacture of bio-pharmaceuticalsand the development of bio-sensors. The ability to produce largequantities of human cells has important working applications for theproduction of human proteins which currently must be obtained fromnon-human sources or donors, implantation of cells to treat diseasessuch as Parkinson's, Alzheimer's and other neurodegenerative diseases;tissues for grafting such as bone marrow, skin, cartilage, tendons,bone, muscle (including cardiac muscle). blood vessels, cornea, neuralcells, gastrointestinal cells and others; and organs for transplantationsuch as kidney, liver, pancreas (including islet cells), heart and lung.

[0181] It is contemplated that multiple different exogenous growthfactors and/or cytokines may be administered in combination with thepolypeptide of the invention to achieve the desired effect, includingany of the growth factors listed herein, other stem cell maintenancefactors, and specifically including stem cell factor (SCF), leukemiainhibitory factor (LIF), Flt-3 ligand (Flt-3L), any, of theinterleukins, recombinant soluble IL-6 receptor fused to IL-6,macrophage inflammatory protein 1-alpha (MIP-1-alpha), G-CSF, GM-CSF,thrombopoietin (TPO), platelet factor 4 (PF-4), platelet-derived growthfactor (PDGF), neural growth factors and basic fibroblast growth factor(bFGF).

[0182] Since totipotent stem cells can give rise to virtually any maturecell type, expansion of these cells in culture will facilitate theproduction of large quantities of mature cells. Techniques for culturingstem cells are known in the art and administration of polypeptides ofthe invention, optionally with other growth factors and/or cytokines, isexpected to enhance the survival and proliferation of the stem cellpopulations. This can be accomplished by direct administration of thepolypeptide of the invention to the culture medium. Alternatively,stroma cells transfected with a polynucleotide that encodes for thepolypeptide of the invention can be used as a feeder layer for the stemcell populations in culture or in vivo. Stromal support cells for feederlayers may include embryonic bone marrow fibroblasts, bone marrowstromal cells, fetal liver cells, or cultured embryonic fibroblasts (seeU.S. Pat. No. 5,690,926).

[0183] Stem cells themselves can be transfected with a polynucleotide ofthe invention to induce autocrine expression of the polypeptide of theinvention. This will allow for generation of undifferentiatedtotipotential/pluripotential stem cell lines that are useful as is orthat can then be differentiated into the desired mature cell types.These stable cell lines can also serve as a source of undifferentiatedtotipotential/pluripotential mRNA to create cDNA libraries and templatesfor polymerase chain reaction experiments. These studies would allow forthe isolation and identification of differentially expressed genes instem cell populations that regulate stem cell proliferation and/ormaintenance.

[0184] Expansion and maintenance of totipotent stem cell populationswill be useful in the treatment of many pathological conditions. Forexample, polypeptides of the present invention may be used to manipulatestem cells in culture to give rise to neuroepithelial cells that can beused to augment or replace cells damaged by illness, autoimmune disease,accidental damage or genetic disorders. The polypeptide of the inventionmay be useful for inducing the proliferation of neural cells and for theregeneration of nerve and brain tissue, i.e. for the treatment ofcentral and peripheral nervous system diseases and neuropathies, as wellas mechanical and traumatic disorders which involve degeneration, deathor trauma to neural cells or nerve tissue. Furthermore, these cells canbe cultured in vitro to form other differentiated cells, such as skintissue that can be used for transplantation. In addition, the expandedstem cell populations can also be genetically altered for gene therapypurposes and to decrease host rejection of replacement tissues aftergrafting or implantation.

[0185] Expression of the polypeptide of the invention and its effect onstem cells can also be manipulated to achieve controlled differentiationof the stem cells into more differentiated cell types. A broadlyapplicable method of obtaining pure populations of a specificdifferentiated cell type from undifferentiated stem cell populationsinvolves the use of a cell-type specific promoter driving a selectablemarker. The selectable marker allows only cells of the desired type tosurvive. For example, stem cells can be induced to differentiate intocardiomyocytes (Wobus et al., Differentiation, 48: 173-182, (1991); Kluget al., J. Clin. Invest., 98(1): 216-224. (1998)) or skeletal musclecells (Browder, L. W. In: Principles of Tissue Engineering eds. Lanza etal., Academic Press (1997)). Alternatively, directed differentiation ofstem cells can be accomplished by culturing the stem cells in thepresence of a differentiation factor such as retinoic acid and anantagonist of the polypeptide of the invention which would inhibit theeffects of endogenous stem cell factor activity and allowdifferentiation to proceed.

[0186] In vitro cultures of stem cells can be used to determine if thepolypeptide of the invention exhibits stem cell growth factor activity.Stem cells are isolated from any one of various cell sources (includinghematopoietic stem cells and embryonic stem cells) and cultured on afeeder layer, as described by Thompson et al, Proc. Natl. Acad. Sci.U.S.A., 9: 7844-7848 (1995), in the presence of the polypeptide of theinvention alone or in combination with other growth factors orcytokines. The ability of the polypeptide of the invention to inducestem cells proliferation is determined by colony formation on semi-solidsupport e.g. as described by Bernstein et al., Blood, 77: 2316-2321(1991).

[0187] 4.7.5 Hematopoiesis Regulating Activity

[0188] A polypeptide of the present invention may be involved inregulation of hematopoiesis and, consequently, in the treatment ofmyeloid or lymphoid cell disorders. Even marginal biological activity insupport of colony forming cells or of factor-dependent cell linesindicates involvement in regulating hematopoiesis, e.g. in supportingthe growth and proliferation of erythroid progenitor cells alone or incombination with other cytokines, thereby indicating utility, forexample, in treating various anemias or for use in conjunction withirradiation/chemotherapy to stimulate the production of erythroidprecursors and/or erythroid cells: in supporting the growth andproliferation of myeloid cells such as granulocytes andmonocytes/macrophages (i.e., traditional colony stimulating factoractivity) useful, for example, in conjunction with chemotherapy toprevent or treat consequent myelo-suppression: in supporting the growthand proliferation of megakaryocytes and consequently of plateletsthereby allowing prevention or treatment of various platelet disorderssuch as thrombocytopenia, and generally for use in place of orcomplimentary to platelet transfusions: and/or in supporting the growthand proliferation of hematopoietic stem cells which are capable ofmaturing to any and all of the above-mentioned hematopoietic cells andtherefore find therapeutic utility in various stem cell disorders (suchas those usually treated with transplantation, including, withoutlimitation, aplastic anemia and paroxysmal nocturnal hemoglobinuria), aswell as in repopulating the stem cell compartment postirradiation/chemotherapy, either in-vivo or ex-vivo (i.e., inconjunction with bone marrow transplantation or with peripheralprogenitor cell transplantation (homologous or heterologous)) as normalcells or genetically manipulated for gene therapy.

[0189] Therapeutic compositions of the invention can be used in thefollowing:

[0190] Suitable assays for proliferation and differentiation of varioushematopoietic lines are cited above.

[0191] Assays for embryonic stem cell differentiation (which willidentify, among others, proteins that influence embryonicdifferentiation hematopoiesis) include, without limitation, thosedescribed in Johansson et al, Cellular Biology 15:141-151, 1995; Kelleret al., Molecular and Cellular Biology 13:473-486, 1993; McClanahan etal., Blood 81:2903-2915, 1993.

[0192] Assays for stem cell survival and differentiation (which willidentify, among others, proteins that regulate lympho-hematopoiesis)include, without limitation, those described in: Methylcellulose colonyforming assays, Freshney, M. G. In Culture of Hematopoietic Cells, R. I.Freshney, et al, eds. Vol pp. 265-268, Wiley-Liss, Inc., New York, N.Y.1994; Hirayama et al., Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992;Primitive hematopoietic colony forming cells with high proliferativepotential, McNiece, I. K, and Briddell, R. A. In Culture ofHematopoietic Cells, R. I. Freshney, et al, eds. Vol pp. 23-39.Wiley-Liss, Inc., New York, N.Y. 1994; Neben et al., ExperimentalHematology 22:353-359, 1994; Cobblestone area forming cell assay.Ploemacher, R. E. In Culture of Hematopoietic Cells, R. I. Freshney, etal, eds. Vol pp. 1-21, Wiley-Liss, Inc., New York, N.Y. 1994; Long termbone marrow cultures in the presence of stromal cells, Spooncer, E.,Dexter, M. and Allen, T. In Culture of Hematopoietic Cells, R. I.Freshney, et al, eds. Vol pp. 163-179, Wiley-Liss, Inc., New York, N.Y.1994; Long term culture initiating cell assay, Sutherland, H. J. InCulture of Hematopoietic Cells, R. I. Freshney, et al, eds. Vol pp.139-162. Wiley-Liss, Inc., New York, N.Y. 1994.

[0193] 4.7.6 Tissue Growth Activity

[0194] A polypeptide of the present invention also may be involved inbone, cartilage tendon, ligament and/or nerve tissue growth orregeneration, as well as in wound healing and tissue repair andreplacement, and in healing of burns, incisions and ulcers.

[0195] A polypeptide of the present invention which induces cartilageand/or bone growth in circumstances where bone is not normally formed,has application in the healing of bone fractures and cartilage damage ordefects in humans and other animals. Compositions of a polypeptide,antibody, binding partner, or other modulator of the invention may haveprophylactic use in closed as well as open fracture reduction and alsoin the improved fixation of artificial joints. De novo bone formationinduced by an osteogenic agent contributes to the repair of congenital,trauma induced, or oncologic resection induced craniofacial defects, andalso is useful in cosmetic plastic surgery.

[0196] A polypeptide of this invention may also be involved inattracting bone-forming cells, stimulating growth of bone-forming cells,or inducing differentiation of progenitors of bone-forming cells.Treatment of osteoporosis, osteoarthritis, bone degenerative disorders,or periodontal disease, such as through stimulation of bone and/orcartilage repair or by blocking inflammation or processes of tissuedestruction (collagenase activity, osteoclast activity, etc.) mediatedby inflammatory processes may also be possible using the composition ofthe invention.

[0197] Another category of tissue regeneration activity that may involvethe polypeptide of the present invention is tendon/ligament formation.Induction of tendon/ligament-like tissue or other tissue formation incircumstances where such tissue is not normally formed, has applicationin the healing of tendon or ligament tears, deformities and other tendonor ligament defects in humans and other animals. Such a preparationemploying a tendon/ligament-like tissue inducing protein may haveprophylactic use in preventing damage to tendon or ligament tissue, aswell as use in the improved fixation of tendon or ligament to bone orother tissues, and in repairing defects to tendon or ligament tissue. Denovo tendon/ligament-like tissue formation induced by a composition ofthe present invention contributes to the repair of congenital, traumainduced, or other tendon or ligament defects of other origin, and isalso useful in cosmetic plastic surgery for attachment or repair oftendons or ligaments. The compositions of the present invention mayprovide environment to attract tendon- or ligament-forming cells,stimulate growth of tendon- or ligament-forming cells, inducedifferentiation of progenitors of tendon- or ligament-forming cells, orinduce growth of tendon/ligament cells or progenitors ex vivo for returnin vivo to effect tissue repair. The compositions of the invention mayalso be useful in the treatment of tendinitis, carpal tunnel syndromeand other tendon or ligament defects. The compositions may also includean appropriate matrix and/or sequestering agent as a carrier as is wellknown in the art.

[0198] The compositions of the present invention may also be useful forproliferation of neural cells and for regeneration of nerve and braintissue, i.e. for the treatment of central and peripheral nervous systemdiseases and neuropathies, as well as mechanical and traumaticdisorders, which involve degeneration, death or trauma to neural cellsor nerve tissue. More specifically, a composition may be used in thetreatment of diseases of the peripheral nervous system, such asperipheral nerve injuries, peripheral neuropathy and localizedneuropathies, and central nervous system diseases, such as Alzheimer's,Parkinson's disease, Huntington's disease, amyotrophic lateralsclerosis, and Shy-Drager syndrome. Further conditions which may betreated in accordance with the present invention include mechanical andtraumatic disorders, such as spinal cord disorders, head trauma andcerebrovascular diseases such as stroke. Peripheral neuropathiesresulting from chemotherapy or other medical therapies may also betreatable using a composition of the invention.

[0199] Compositions of the invention may also be useful to promotebetter or faster closure of non-healine wounds, including withoutlimitation pressure ulcers, ulcers associated with vascularinsufficiency, surgical and traumatic wounds, and the like.

[0200] Compositions of the present invention may also be involved in thegeneration or regeneration of other tissues, such as organs (including,for example, pancreas, liver, intestine, kidney, skin, endothelium),muscle (smooth, skeletal or cardiac) and vascular (including vascularendothelium) tissue, or for promoting the growth of cells comprisingsuch tissues. Part of the desired effects may be by inhibition ormodulation of fibrotic scarring may allow normal tissue to regenerate. Apolypeptide of the present invention may also exhibit angiogenicactivity.

[0201] A composition of the present invention may also be useful for gutprotection or regeneration and treatment of lung or liver fibrosis,reperfusion injury in various tissues, and conditions resulting fromsystemic cytokine damage.

[0202] A composition of the present invention may also be useful forpromoting or inhibiting differentiation of tissues described above fromprecursor tissues or cells: or for inhibiting the growth of tissuesdescribed above.

[0203] Therapeutic compositions of the invention can be used in thefollowing:

[0204] Assays for tissue generation activity include, withoutlimitation, those described in: International Patent Publication No.WO95/16035 (bone, cartilage, tendon): International Patent PublicationNo. WO95/05846 (nerve, neuronal): International Patent Publication No.WO91/07491 (skin, endothelium).

[0205] Assays for wound healings activity include, without limitation,those described in: Winter, Epidermal Wound Healing, pp. 71-112(Maibach, H. I. and Rovee, D. T., eds.). Year Book Medical Publishers,Inc., Chicago, as modified by Eaglstein and Mertz, J. Invest, Dermatol71:382-84 (1978).

[0206] 4.7.7 Immune Function Stimulating or Suppressing Activity

[0207] A polypeptide of the present invention may also exhibit immunestimulating or immune suppressing activity, including without limitationthe activities for which assays are described herein. A polynucleotideof the invention can encode a polypeptide exhibiting such activities. Aprotein may be useful in the treatment of various immune deficienciesand disorders (including severe combined immunodeficiency (SCID)), e.g.,in regulating (up or down) growth and proliferation of T and/or Blymphocytes, as well as effecting the cytolytic activity of NK cells andother cell populations. These immune deficiencies may be genetic or becaused by viral (e.g., HIV) as well as bacterial or fungal infections,or may result from autoimmune disorders, more specifically, infectiousdiseases causes by viral, bacterial, fungal or other infection may betreatable using a protein of the present invention, including infectionsby HIV, hepatitis viruses, herpes viruses, mycobacteria. Leishmaniaspp., malaria spp, and various fungal infections such as candidiasis. Ofcourse, in this regard, proteins of the present invention may also beuseful where a boost to the immune system generally may be desirable,i.e., in the treatment of cancer.

[0208] Autoimmune disorders which may be treated using a protein of thepresent invention include, for example, connective tissue disease,multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis,autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmunethyroiditis, insulin dependent diabetes mellitis, myasthenia gravis,graft-versus-host disease and autoimmune inflammatory eye disease. Sucha protein (or antagonists thereof, including antibodies) of the presentinvention may also to be useful in the treatment of allergic reactionsand conditions (e.g., anaphylaxis, serum sickness, drug reactions, foodallergies, insect venom allergies, mastocytosis, allergic rhinitis,hypersensitivity pneumonitis, urticaria, angioedema, eczema, atopicdermatitis, allergic contact dermatitis, erythema multiforme,Stevens-Johnson syndrome, allergic conjunctivitis, atopickeratoconjunctivitis, venereal keratoconjunctivitis, giant papillaryconjunctivitis and contact allergies), such as asthma (particularlyallergic asthma) or other respiratory problems. Other conditions, inwhich immune suppression is desired (including, for example, organtransplantation), may also be treatable using a protein (or antagoniststhereof) of the present invention. The therapeutic effects of thepolypeptides or antagonists thereof on allergic reactions can beevaluated by in vivo animals models such as the cumulative contactenhancement test (Lastbom et al., Toxicology 125: 59-66, 1998), skinprick test (Hoffmann et al., Allergy 54: 446-54, 1999), guinea pig skinsensitization test (Vohr et al., Arch. Toxocol. 73: 501-9), and murinelocal lymph node assay (Kimber et al., J. Toxicol. Environ. Health 53:563-79).

[0209] Using the proteins of the invention it may also be possible tomodulate immune responses, in a number of ways. Down regulation may bein the form of inhibiting or blocking an immune response already inprogress or may involve preventing the induction of an immune response.The functions of activated T cells may be inhibited by suppressing Tcell responses or by inducing specific tolerance in T cells, or both.Immunosuppression of T cell responses is generally an active,non-antigen-specific, process which requires continuous exposure of theT cells to the suppressive agent. Tolerance, which involves inducingnon-responsiveness or anergy in T cells, is distinguishable fromimmunosuppression in that it is generally antigen-specific and persistsafter exposure to the tolerizing agent has ceased. Operationally,tolerance can be demonstrated by the lack of a T cell response uponreexposure to specific antigen in the absence of the tolerizing agent.

[0210] Down regulating or preventing one or more antigen functions(including without limitation B lymphocyte antigen functions (such as,for example, B7)), e.g., preventing high level lymphokine synthesis byactivated T cells, will be useful in situations of tissue, skin andorgan transplantation and in graft-versus-host disease (GVHD). Forexample, blockage of T cell function should result in reduced tissuedestruction in tissue transplantation. Typically, in tissue transplants,rejection of the transplant is initiated through its recognition asforeign by T cells, followed by an immune reaction that destroys thetransplant. The administration of a therapeutic composition of theinvention may prevent cytokine synthesis by immune cells, such as Tcells, and thus acts as an immunosuppressant. Moreover, a lack ofcostimulation may also be sufficient to anergize the T cells, therebyinducing tolerance in a subject. Induction of long-term tolerance by Blymphocyte antigen-blocking reagents may avoid the necessity of repeatedadministration of these blocking reagents. To achieve sufficientimmunosuppression or tolerance in a subject, it may also be necessary toblock the function of a combination of B lymphocyte antigens.

[0211] The efficacy of particular therapeutic compositions in preventingorgan transplant rejection or GVHD can be assessed using animal modelsthat are predictive of efficacy in humans. Examples of appropriatesystems which can be used include allogeneic cardiac grafts in rats andxenogeneic pancreatic islet cell grafts in mice, both of which have beenused to examine the immunosuppressive effects of CTLA4Ig fusion proteinsin vivo as described in Lenschow et al., Science 257:789-792 (1992) andTurka et al., Proc. Natl. Acad. Sci USA, 89:11102-11105 (1992). Inaddition, murine models of GVHD (see Paul ed., Fundamental Immunology,Raven Press, New York, 1989, pp. 846-847) can be used to determine theeffect of therapeutic compositions of the invention on the developmentof that disease.

[0212] Blocking antigen function may also be therapeutically useful fortreating autoimmune diseases. Many autoimmune disorders are the resultof inappropriate activation of T cells that are reactive against selftissue and which promote the production of cytokines and autoantibodiesinvolved in the pathology of the diseases. Preventing the activation ofautoreactive T cells may reduce or eliminate disease symptoms.Administration of reagents which block stimulation of T cells can beused to inhibit T cell activation and prevent production ofautoantibodies or T cell-derived cytokines which may be involved in thedisease process. Additionally, blocking reagents may induceantigen-specific tolerance of autoreactive T cells which could lead tolong-term relief from the disease. The efficacy of blocking reagents inpreventing or alleviating autoimmune disorders can be determined using anumber of well-characterized animal models of human autoimmune diseases.Examples include murine experimental autoimmune encephalitis, systemiclupus erythematosus in MRL/lpr/lpr mice or NZB hybrid mice, murineautoimmune collagen arthritis, diabetes mellitus in NOD mice and BBrats, and murine experimental myasthenia gravis (see Paul ed.,Fundamental Immunology, Raven Press, New York, 1989, pp. 840-856).

[0213] Upregulation of an antigen function (e.g., a B lymphocyte antigenfunction), as a means of up regulating immune responses, may also beuseful in therapy. Upregulation of immune responses may be in the formof enhancing an existing immune response or eliciting an initial immuneresponse. For example, enhancing an immune response may be useful incases of viral infection, including systemic viral diseases such asinfluenza, the common cold, and encephalitis.

[0214] Alternatively, anti-viral immune responses may be enhanced in aninfected patient by removing T cells from the patient, costimulating theT cells in vitro with viral antigen-pulsed APCs either expressing apeptide of the present invention or together with a stimulatory form ofa soluble peptide of the present invention and reintroducing the invitro activated T cells into the patient. Another method of enhancinganti-viral immune responses would be to isolate infected cells from apatient, transfect them with a nucleic acid encoding a protein of thepresent invention as described herein such that the cells express all ora portion of the protein on their surface, and reintroduce thetransfected cells into the patient. The infected cells would now becapable of delivering a costimulatory signal to, and thereby activate, Tcells in vivo.

[0215] A polypeptide of the present invention may provide the necessarystimulation signal to T cells to induce a T cell mediated immuneresponse against the transfected tumor cells. In addition, tumor cellswhich lack MHC class I or MHC class II molecules, or which fail toreexpress sufficient mounts of MHC class I or MHC class II molecules,can be transfected with nucleic acid encoding all or a portion of (e.g.,a cytoplasmic-domain truncated portion) of an MHC class I alpha chainprotein and β₂ microglobulin protein or an MHC class II alpha chainprotein and an MHC class II beta chain protein to thereby express MHCclass I or MHC class II proteins on the cell surface. Expression of theappropriate class I or class II MHC in conjunction with a peptide havingthe activity of a B lymphocyte antigen (e.g., B7-1, B7-2, B7-3) inducesa T cell mediated immune response against the transfected tumor cell.Optionally, a gene encoding an antisense construct which blocksexpression of an MHC class II associated protein, such as the invariantchain, can also be cotransfected with a DNA encoding a peptide havingthe activity of a B lymphocyte antigen to promote presentation of tumorassociated antigens and induce tumor specific immunity. Thus, theinduction of a T cell mediated immune response in a human subject may besufficient to overcome tumor-specific tolerance in the subject.

[0216] The activity of a protein of the invention may, among othermeans, be measured by the following methods:

[0217] Suitable assays for thymocyte or splenocyte cytotoxicity include,without limitation, those described in: Current Protocols in Immunology,Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Maroulies. E. M. Shevach, W.Strober, Pub. Greene Publishing Associates and Wiley-Interscience(Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19:Chapter 7, Immunologic studies in Humans): Herrmann et al., Proc. Natl.Acad. Sci. USA 78:2488-2492, 1981; Herrmann et al., J. Immunol.128:1968-1974, 1982; Handa et al., J. Immunol. 135:1564-1572, 1985;Takai et al., I. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol.140:508-512, 1988; Bowman et al., J. Virology 61:199-1998; Benagnolli etal., Cellular Immunology 133:327-341, 1991; Brown et al., J. Immunol.153:3079-3092,1994.

[0218] Assays for T-cell-dependent immunoglobulin responses and isotypeswitching (which will identify, among others, proteins that modulateT-cell dependent antibody responses and that affect Th1/Th2 profiles)include, without limitation, those described in: Maliszewski, J.Immunol. 144:3028-3033, 1990; and Assays for B cell function: In vitroantibody production, Mond, J. J, and Brunswick, M. In Current Protocolsin Immunology, J. E. e.a. Coligan eds. Vol 1 pp. 3.8.1-3.8.16, JohnWiley and Sons, Toronto, 1994.

[0219] Mixed lymphocyte reaction (MLR) assays (which will identify,among others, proteins that generate predominantly Th1 and CTLresponses) include, without limitation, those described in: CurrentProtocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H.Margulies, E. M. Shevach, W. Strober, Pub. Greene Publishing Associatesand Wiley-Interscience (Chapter 3, In Vitro assays for Mouse LymphocyteFunction 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai etal., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol.140:508-512, 1988; Bertagnolli et al., J. Immunol. 149:3778-3783, 1992.

[0220] Dendritic cell-dependent assays (which will identify, amongothers, proteins expressed by dendritic cells that activate naiveT-cells) include, without limitation, those described in: Guery et al.,J. Immunol. 134:536-544, 1995; Inaba et al., Journal of ExperimentalMedicine 173:549-559, 1991; Macatonia et al., Journal of Immunology154:5071-5079, 1995; Porgador et al., Journal of Experimental Medicine182:255-260, 1995; Nair et al., Journal of Virology 67:4062-4069, 1993;Huang et al., Science 264:961-965, 1994: Macatonia et al., Journal ofExperimental Medicine 169:1255-1264, 1989; Bhardwaj et al., Journal ofClinical Investigation 94:797-807, 1994; and Inaba et al., Journal ofExperimental Medicine 172:631-640, 1990.

[0221] Assays for lymphocyte survival/apoptosis (which will identify,among others, proteins that prevent apoptosis after superantigeninduction and proteins that regulate lymphocyte homeostasis) include,without limitation, those described in: Darzynkiewicz et al., Cytometry13:795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993; Gorczyca etal., Cancer Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243,1991; Zacharchuk, Journal of Immunology 145:4037-4045, 1990; Zamai etal., Cytometry 14:891-897, 1993; Gorczyca et al., International Journalof Oncology 1:639-648, 1992.

[0222] Assays for proteins that influence early steps of T-cellcommitment and development include, without limitation, those describedin: Antica et al., Blood 84:111-117, 1994; Fine et al., CellularImmunology 155:111-122, 1994; Galy et al., Blood 85:2770-2778, 1995;Toki et al., Proc. Nat. Acad Sci. USA 88:7548-7551, 1991.

[0223] 4.7.8 Chemotactic/Chemokinetic Activity

[0224] A polypeptide of the present invention may be involved inchemotactic or chemokinetic activity for mammalian cells, including, forexample, monocytes, fibroblasts, neutrophils, T-cells, mast cells,eosinophils, epithelial and/or endothelial cells. A polynucleotide ofthe invention can encode a polypeptide exhibiting such attributes.Chemotactic and chemokinetic receptor activation can be used to mobilizeor attract a desired cell population to a desired site of action.Chemotactic or chemokinetic compositions (e.g. proteins, antibodies,binding partners, or modulators of the invention) provide particularadvantages in treatment of wounds and other trauma to tissues, as wellas in treatment of localized infections. For example, attraction oflymphocytes, monocytes or neutrophils to tumors or sites of infectionmay result in improved immune responses against the tumor or infectingagent.

[0225] A protein or peptide has chemotactic activity for a particularcell population if it can stimulate, directly or indirectly, thedirected orientation or movement of such cell population. Preferably,the protein or peptide has the ability to directly stimulate directedmovement of cells. Whether a particular protein has chemotactic activityfor a population of cells can be readily determined by employing suchprotein or peptide in any known assay for cell chemotaxis.

[0226] Therapeutic compositions of the invention can be used in thefollowing:

[0227] Assays for chemotactic activity (which will identify proteinsthat induce or prevent chemotaxis) consist of assays that measure theability of a protein to induce the migration of cells across a membraneas well as the ability of a protein to induce the adhesion of one cellpopulation to another cell population. Suitable assays for movement andadhesion include, without limitation, those described in: CurrentProtocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H.Marguiles, E. M. Shevach, W. Strober, Pub. Greene Publishing Associatesand Wiley-Interscience (Chapter 6.12, Measurement of alpha and betaChemokines 6.12.1-6.12.28; Taub et al. J. Clin. Invest. 95:1370-1376,1995; Lind et al, APMIS 103:140-146, 1995; Muller et al Eur. J. Immunol.25:1744-1748: Gruber et al. J. of Immunol, 152:5860-5867, 1994; Johnstonet al, J. of Immunol. 153:1762-1768, 1994.

[0228] 4.7.9 Hemostatic and Thrombolytic Activity

[0229] A polypeptide of the invention may also be involved in hemostatisor thrombolysis or thrombosis. A polynucleotide of the invention canencode a polypeptide exhibiting such attributes. Compositions may beuseful in treatment of various coagulation disorders (includinghereditary disorders, such as hemophilias) or to enhance coagulation andother hemostatic events in treating wounds resulting from trauma,surgery or other causes. A composition of the invention may also beuseful for dissolving or inhibiting formation of thromboses and fortreatment and prevention of conditions resulting therefrom (such as, forexample, infarction of cardiac and central nervous system vessels (e.g.,stroke).

[0230] Therapeutic compositions of the invention can be used in thefollowing:

[0231] Assay for hemostatic and thrombolytic activity include withoutlimitation, those described in: Linet et al., J. Clin. Pharmacol.26:131-140, 1986; Burdick et al., Thrombosis Res. 45:413-419, 1987;Humphrey et al., Fibrinolysis 5:71-79 (1991); Schaub, Prostaglandins35:467-474, 1988.

[0232] 4.7.10 Cancer Diagnosis and Therapy

[0233] Polypeptides of the invention may be involved in cancer cellgeneration, proliferation or metastasis. Detection of the presence oramount of polynucleotides or polypeptides of the invention may be usefulfor the diagnosis and/or prognosis of one or more types of cancer. Forexample, the presence or increased expression of apolynucleotide/polypeptide of the invention may indicate a hereditaryrisk of cancer, a precancerous condition, or an ongoing malignancy.Conversely, a defect in the gene or absence of the polypeptide may beassociated with a cancer condition. Identification of single nucleotidepolymorphisms associated with cancer or a predisposition to cancer mayalso be useful for diagnosis or prognosis.

[0234] Cancer treatments promote tumor regression by inhibiting tumorcell proliferation, inhibiting angiogenesis (growth of new blood vesselsthat is necessary to support tumor growth) and/or prohibiting metastasisby reducing tumor cell motility or invasiveness. Therapeuticcompositions of the invention may be effective in adult and pediatriconcology including in solid phase tumors/malignancies, locally advancedtumors, human soft tissue sarcomas, metastatic cancer, includinglymphatic metastases, blood cell malignancies including multiplemyeloma, acute and chronic leukemias, and lymphomas, head and neckcancers including mouth cancer, larynx cancer and thyroid cancer, lungcancers including small cell carcinoma and non-small cell cancers,breast cancers including small cell carcinoma and ductal carcinoma,gastrointestinal cancers including esophageal cancer, stomach cancer,colon cancer, colorectal cancer and polyps associated with colorectalneoplasia, pancreatic cancers, liver cancer, urologic cancers includingbladder cancer and prostate cancer, malignancies of the female genitaltract including ovarian carcinoma, uterine (including endometrial)cancers, and solid tumor in the ovarian follicle, kidney cancersincluding renal cell carcinoma, brain cancers including intrinsic braintumors, neuroblastoma, astrocytic brain tumors, gliomas, metastatictumor cell invasion in the central nervous system, bone cancersincluding osteomas, skin cancers including malignant melanoma, tumorprogression of human skin keratinocytes, squamous cell carcinoma, basalcell carcinoma, hemangiopericytoma and Karposi's sarcoma.

[0235] Polypeptides, polynucleotides, or modulators of polypeptides ofthe invention (including inhibitors and stimulators of the biologicalactivity of the polypeptide of the invention) may be administered totreat cancer. Therapeutic compositions can be administered intherapeutically effective dosages alone or in combination with adjuvantcancer therapy such as surgery, chemotherapy, radiotherapy,thermotherapy, and laser therapy, and may provide a beneficial effect,e.g. reducing tumor size, slowing rate of tumor growth, inhibitingmetastasis, or otherwise improving overall clinical condition, withoutnecessarily eradicating the cancer.

[0236] The composition can also be administered in therapeuticallyeffective amounts as a portion of an anti-cancer cocktail. Ananti-cancer cocktail is a mixture of the polypeptide or modulator of theinvention with one or more anti-cancer drugs in addition to apharmaceutically acceptable carrier for delivery. The use of anti-cancercocktails as a cancer treatment is routine. Anti-cancer drugs that arewell known in the art and can be used as a treatment in combination withthe polypeptide or modulator of the invention include: Actinomycin D,Aminoglutethimide, Asparaginase, Bleomycin, Busulfan, Carboplatin,Carmustine, Chlorambucil, Cisplatin (cis-DDP), Cyclophosphamide,Cytarabine HCl (Cytosine arabinoside), Dacarbazine, Dactinomycin,Daunorubicin HCl, Doxorubicin HCl, Estramustine phosphate sodium,Etoposide (V16-213), Floxuridine, 5-Fluorouracil (5-Fu), Flutamide,Hydroxyurea (hydroxycarbamide), Ifosfamide, Interferon Alpha-2a,Interferon Alpha-2b, Leuprolide acetate (LHRH-releasing factor analog),Lomustine, Mechlomorethamine HCl (nitrogen mustard), Melphalan,Mercaptopurine, Mesna, Methotrexate (MTX), Mitomycin, Mitoxantrone HCl,Octreotide, Plicamycin, Procarbazine HCl, Streptozocin, Tamoxifencitrate, Thioguanine, Thiotepa, Vinblastine sulfate, Vincristinesulfate, Amsacrine, Azacitidine, Hexamethylmelamine, Interleukin-2,Mitoguazone, Pentostatin, Semustine, Teniposide, and Vindesine sulfate.

[0237] In addition, therapeutic compositions of the invention may beused for prophylactic treatment of cancer. There are hereditaryconditions and/or environmental situations (e.g. exposure tocarcinogens) known in the art that predispose an individual todeveloping cancers. Under these circumstances, it may be beneficial totreat these individuals with therapeutically effective doses of thepolypeptide of the invention to reduce the risk of developing cancers.

[0238] In vitro models can be used to determine the effective doses ofthe polypeptide of the invention as a potential cancer treatment. Thesein vitro models include proliferation assays of cultured tumor cells,growth of cultured tumor cells in soft agar (see Freshney, (1987)Culture of Animal Cells: A Manual of Basic Technique, Wily-Liss, NewYork, N.Y. Ch 18 and Ch 21). tumor systems in nude mice as described inGiovanella et al., J. Natl. Can. Inst., 52: 921-30 (1974), mobility andinvasive potential of tumor cells in Boyden Chamber assays as describedin Pilkington et al., Anticancer Res., 17: 4107-9 (1997), andangiogenesis assays such as induction of vascularization of the chickchorioallantoic membrane or induction of vascular endothelial cellmigration as described in Ribatta et al., Intl. J. Dev. Biol., 40:1189-97 (1999) and Li et al., Clin. Exp. Metastasis, 17:423-9(1999),respectively. Suitable tumor cells lines are available, e.g. fromAmerican Type Tissue Culture Collection catalogs.

[0239] 4.7.11 Receptor/Ligand Activity

[0240] A polypeptide of the present invention may also demonstrateactivity as receptor, receptor ligand or inhibitor or agonist ofreceptor/ligand interactions. A polynucleotide of the invention canencode a polypeptide exhibiting such characteristics, Examples of suchreceptors and ligands include, without limitation, cytokine receptorsand their ligands, receptor kinases and their ligands, receptorphosphatases and their ligands, receptors involved in cell-cellinteractions and their ligands (including without limitation, cellularadhesion molecules (such as selectins, integrins and their ligands) andreceptor/ligand pairs involved in antigen presentation, antigenrecognition and development of cellular and humoral immune responses.Receptors and ligands are also useful for screening of potential peptideor small molecule inhibitors of the relevant receptor/ligandinteraction. A protein of the present invention (including, withoutlimitation, fragments of receptors and ligands) may themselves be usefulas inhibitors of receptor/ligand interactions.

[0241] The activity of a polypeptide of the invention may, among othermeans, be measured by the following methods:

[0242] Suitable assays for receptor-ligand activity include withoutlimitation those described in: Current Protocols in Immunology, Ed by J.E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober,Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 7.28.Measurement of Cellular Adhesion under static conditions7.28.1-7.28.22), Takai et al., Proc. Natl. Acad. Sci. USA 84:6864-6868,1987; Bierer et al., J. Exp. Med. 168:1145-1156, 1988; Rosenstein etal., J. Exp. Med. 169:149-160 1989; Stoltenborg et al., J. Immunol.Methods 175:59-68, 1994; Stitt et al., Cell 80:661-670, 1995.

[0243] By way of example, the polypeptides of the invention may be usedas a receptor for a ligand(s) thereby transmitting the biologicalactivity of that ligand(s). Ligands may be identified throuh bindingassays, affinity chromatography, dihybrid screening assays. BIAcoreassays, gel overlay assays, or other methods known in the art.

[0244] Studies characterizing drugs or proteins as agonist or antagonistor partial agonists or a partial antagonist require the use of otherproteins as competing ligands. The polypeptides of the present inventionor ligand(s) thereof may be labeled by being coupled to radioisotopes,colorimetric molecules or a toxin molecules by conventional methods.(“Guide to Protein Purification” Murray P. Deutscher (ed) Methods inEnzymology Vol. 182 (1990) Academic Press. Inc. San Diego). Examples ofradioisotopes include, but are not limited to, tritium and carbon-14.Examples of colorimetric molecules include, but are not limited to,fluorescent molecules such as fluorescamine, or rhodamine or othercolorimetric molecules. Examples of toxins include, but are not limited,to ricin.

[0245] 4.7.12 Drug Screening

[0246] This invention is particularly useful for screening chemicalcompounds by using the novel polypeptides or binding fragments thereofin any of a variety of drug screening techniques. The polypeptides orfragments employed in such a test may either be free in solution,affixed to a solid support, borne on a cell surface or locatedintracellularly. One method of drug screening utilizes eukaryotic orprokaryotic host cells which are stably transformed with recombinantnucleic acids expressing the polypeptide or a fragment thereof. Drugsare screened against such transformed cells in competitive bindingassays. Such cells, either in viable or fixed form, can be used forstandard binding assays. One may measure, for example, the formation ofcomplexes between polypeptides of the invention or fragments and theagent being tested or examine the diminution in complex formationbetween the novel polypeptides and an appropriate cell line, which arewell known in the art.

[0247] Sources for test compounds that may be screened for ability tobind to or modulate (i.e., increase or decrease) the activity ofpolypeptides of the invention include (1) inorganic and organic chemicallibraries. (2) natural product libraries, and (3) combinatoriallibraries comprised of either random or mimetic peptides,oligonucleotides or organic molecules.

[0248] Chemical libraries may be readily synthesized or purchased from anumber of commercial sources, and may include structural analogs ofknown compounds or compounds that are identified as “hits” or “leads”via natural product screening.

[0249] The sources of natural product libraries are microorganisms(including bacteria and fungi), animals, plants or other vegetation, ormarine organisms, and libraries of mixtures for screening may be createdby: (1) fermentation and extraction of broths from soil, plant or marinemicroorganisms or (2) extraction of the organisms themselves. Naturalproduct libraries include polyketides, non-ribosomal peptides, and(non-naturally occurring) variants thereof. For a review, see Science282:63-68 (1998).

[0250] Combinatorial libraries are composed of large numbers ofpeptides, oligonucleotides or organic compounds and can be readilyprepared by traditional automated synthesis methods. PCR, cloning orproprietary synthetic methods. Of particular interest are peptide andoligonucleotide combinatorial libraries. Still other libraries ofinterest include peptide, protein, peptidomimetic, multiparallelsynthetic collection, recombinatorial, and polypeptide libraries. For areview of combinatorial chemistry and libraries created therefrom, seeMyers. Curr. Opin. Biotechnol. 8:701-707 (1997). For reviews andexamples of peptidomimetic libraries, see Al-Obeidi et al., Mol.Biotechnol. 9(3):205-23 (1998); Hruby et al., Curr Opin Chem Biol. 1(1):114-19 (1997): Dorner et al., Bioorg Med Chem. 4(5):709-15 (1996)(alkylated dipeptides).

[0251] Identification of modulators through use of the various librariesdescribed herein permits modification of the candidate “hit” (or “lead”)to optimize the capacity of the “hit” to bind a polypeptide of theinvention. The molecules identified in the binding assay are then testedfor antagonist or agonist activity in in vivo tissue culture or animalmodels that are well known in the art. In brief, the molecules aretitrated into a plurality of cell cultures or animals and then testedfor either cell/animal death or prolonged survival of the animal/cells.

[0252] The binding molecules thus identified may be complexed withtoxins, e.g., ricin or cholera, or with other compounds that are toxicto cells such as radioisotopes. The toxin-binding molecule complex isthen targeted to a tumor or other cell by the specificity of the bindingmolecule for a polypeptide of the invention. Alternatively, the bindingmolecules may be complexed with imaging agents for targeting and imagingpurposes.

[0253] 4.7.13 Assay for Receptor Activity

[0254] The invention also provides methods to detect specific binding ofa polypeptide e.g. a ligand or a receptor. The art provides numerousassays particularly useful for identifying previously unknown bindingpartners for receptor polypeptides of the invention. For example,expression cloning using mammalian or bacterial cells, or dihybridscreening assays can be used to identify polynucleotides encodingbinding partners. As another example, affinity chromatography with theappropriate immobilized polypeptide of the invention can be used toisolate polypeptides that recognize and bind polypeptides of theinvention. There are a number of different libraries used for theidentification of compounds, and in particular small molecules, thatmodulate (i.e., increase or decrease) biological activity of apolypeptide of the invention. Ligands for receptor polypeptides of theinvention can also be identified by adding exogenous ligands, orcocktails of ligands to two cells populations that are geneticallyidentical except for the expression of the receptor of the invention:one cell population expresses the receptor of the invention whereas theother does not. The response of the two cell populations to the additionof ligands(s) are then compared. Alternatively, an expression librarycan be co-expressed with the polypeptide of the invention in cells andassayed for an autocrine response to identify potential ligand(s). Asstill another example. BIAcore assays, gel overlay assays, or othermethods known in the art can be used to identify binding partnerpolypeptides, including, (1) organic and inorganic chemical libraries(2) natural product libraries, and (3) combinatorial libraries comprisedof random peptides, oligonucleotides or organic molecules.

[0255] The role of downstream intracellular signaling molecules in thesignaling cascade of the polypeptide of the invention can be determined.For example, a chimeric protein in which the cytoplasmic domain of thepolypeptide of the invention is fused to the extracellular portion of aprotein, whose ligand has been identified, is produced in a host cell.The cell is then incubated with the ligand specific for theextracellular portion of the chimeric protein, thereby activating thechimeric receptor. Known downstream proteins involved in intracellularsignaling can then be assayed for expected modifications i.e.phosphorylation. Other methods known to those in the art can also beused to identify signaling molecules involved in receptor activity.

[0256] 4.7.14 Leukemias

[0257] Leukemias and related disorders may be treated or prevented byadministration of a therapeutic that promotes or inhibits function ofthe polynucleotides and/or polypeptides of the invention. Such leukemiasand related disorders include but are not limited to acute leukemia,acute lymphocytic leukemia, acute myelocytic leukemia, myeloblastic,promyelocytic, myelomonocytic, monocytic, erythroleukemia, chronicleukemia, chronic myelocytic (granulocytic) leukemia and chroniclymphocytic leukemia (for a review of such disorders, see Fishman etal., 1985, Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia).

[0258] 4.7.15 Nervous System Disorders

[0259] Nervous system disorders involving cell types which can be testedfor efficacy of intervention with compounds that modulate the activityof the polynucleotides and/or polypeptides of the invention, and whichcan be treated upon thus observing an indication of therapeutic utility,include but are not limited to nervous system injuries, and diseases ordisorders which result in either a disconnection of axons, a diminutionor degeneration of neurons, or demyelination. Nervous system lesionswhich may be treated in a patient (including human and non-humanmammalian patients) according to the invention include but are notlimited to the following lesions of either the central (including spinalcord, brain) or peripheral nervous systems:

[0260] (i) traumatic lesions, including lesions caused by physicalinjury or associated with surgery, for example, lesions which sever aportion of the nervous system, or compression injuries:

[0261] (ii) ischemic lesions in which a lack of oxygen in a portion ofthe nervous system results in neuronal injury or death, includingcerebral infarction or ischemia, or spinal cord infarction or ischemia:

[0262] (iii) infectious lesions, in which a portion of the nervoussystem is destroyed or injured as a result of infection, for example, byan abscess or associated with infection by human immunodeficiency virus,herpes zoster, or herpes simplex virus or with Lyme disease,tuberculosis, syphilis:

[0263] (iv) degenerative lesions in which a portion of the nervoussystem is destroyed or injured as a result of a degenerative processincluding but not limited to degeneration associated with Parkinson'sdisease, Alzheimer's disease, Huntington's chorea, or amyotrophiclateral sclerosis;

[0264] (v) lesions associated with nutritional diseases or disorders, inwhich a portion of the nervous system is destroyed or injured by anutritional disorder or disorder of metabolism including but not limitedto vitamin B12 deficiency, folic acid deficiency, Wernicke disease,tobacco-alcohol amblyopia. Marchiafava-Bignami disease (primarydegeneration of the corpus callosum), and alcoholic cerebellardegeneration:

[0265] (vi) neurological lesions associated with systemic diseasesincluding but not limited to diabetes (diabetic neuropathn. Bell'spalsy), systemic lupus erythematosus carcinoma, or sarcoidosis;

[0266] (vii) lesions caused by toxic substances including alcohol, lead,or particular neurotoxins; and

[0267] (viii) demyelinated lesions in which a portion of the nervoussystem is destroyed or injured by a demyelinating disease including butnot limited to multiple sclerosis, human immunodeficiencyvirus-associated myelopathy, transverse myelopathy or variousetiologies. progressive multifocal leukoencephalopathy, and centralpontine myelinolysis.

[0268] Therapeutics which are useful according to the invention fortreatment of a nervous system disorder may be selected by testing forbiological activity in promoting the survival or differentiation ofneurons. For example, and not by way of limitation, therapeutics whichelicit any of the following effects may be useful according to theinvention:

[0269] (i) increased survival time of neurons in culture;

[0270] (ii) increased sprouting of neurons in culture or in vivo;

[0271] (iii) increased production of a neuron-associated molecule inculture or in vivo, e.g. choline acetyltransferase oracetylcholinesterase with respect to motor neurons; or

[0272] (iv) decreased symptoms of neuron dysfunction in vivo.

[0273] Such effects may be measured by any method known in the art. Inpreferred non-limiting embodiments, increased survival of neurons may bemeasured by the method set forth in Arakaawa et al. (1990. J. Neurosci.10:3507-3515): increased sprouting of neurons may be detected by methodsset forth in Pestronk et al. (1980. Exp. Neurol. 70:6-82) or Brown etal. (1981. Ann. Rev. Neurosci. 4:17-42); increased production ofneuron-associated molecules may be measured by bioassay, enzymaticassay, antibody binding, Northern blot assay, etc., depending on themolecule to be measured; and motor neuron dysfunction man be measured byassessing the physical manifestation of motor neuron disorder, e.g.,weakness, motor neuron conduction velocity, or functional disability.

[0274] In specific embodiments, motor neuron disorders that may betreated according to the invention include but are not limited todisorders such as infarction, infection, exposure to toxin, trauma,surgical damage, degenerative disease or malignancy that may affectmotor neurons as well as other components of the nervous system, as wellas disorders that selectively affect neurons such as amyotrophic lateralsclerosis, and including but not limited to progressive spinal muscularatrophy, progressive bulbar palsy, primary lateral sclerosis, infantileand juvenile muscular atrophy, progressive bulbar paralysis of childhood(Fazio-Londe syndrome), poliomyelitis and the post polio syndrome, andHereditary Motorsensory Neuropathy (Charcot-Marie-Tooth Disease).

[0275] 4.7.16 Other Activities

[0276] A polypeptide of the invention may also exhibit one or more ofthe following additional activities or effects: inhibiting the growth,infection or function of, or killing, infectious agents, including,without limitation, bacteria, viruses, fungi, and other parasites;effecting (suppressing or enhancing) bodily characteristics, including,without limitation, height, weight, hair color, eye color, skin, fat tolean ratio or other tissue pigmentation, or organ or body part size orshape (such as, for example, breast augmentation or diminution, changein bone form or shape); effecting biorhythms or circadian cycles orrhythms; effecting the fertility of male or female subjects; effectingthe metabolism, catabolism, anabolism processing, utilization, storageor elimination of dietary fat, lipid, protein, carbohydrate, vitamins,minerals, co-factors or other nutritional factors or component(s);effecting behavioral characteristics, including, without limitation,appetite, libido, stress, cognition (including cognitive disorders),depression (including depressive disorders) and violent behaviors;providing analgesic effects or other pain reducing effects; promotingdifferentiation and growth of embryonic stem cells in lineages otherthan hematopoietic lineages; hormonal or endocrine activity; in the caseof enzymes, correcting deficiencies of the enzyme and treatingdeficiency-related diseases; treatment of hyperproliferative disorders(such as, for example, psoriasis); immunoglobulin-like activity (suchas, for example, the ability to bind antigens or complement); and theability to act as an antigen in a vaccine composition to raise an immuneresponse against such protein or another material or entity which iscross-reactive with such protein.

[0277] 4.7.17 Identification of Polymorphisms

[0278] The demonstration of polymorphisms makes possible theidentification of such polymorphisms in human subjects and thepharmacogenetic use of this information for diagnosis and treatment.Such polymorphisms may be associated with, e.g., differentialpredisposition or susceptibility to various disease states (such asdisorders involving inflammation or immune response) or a differentialresponse to drug administration, and this genetic information can beused to tailor preventive or therapeutic treatment appropriately. Forexample, the existence of a polymorphism associated with apredisposition to inflammation or autoimmune disease makes possible thediagnosis of this condition in humans by identifying the presence of thepolymorphism.

[0279] Polymorphisms can be identified in a variety of ways known in theart which all generally involve obtaining a sample from a patient,analyzing DNA from the sample, optionally involving isolation oramplification of the DNA, and identifying the presence of thepolymorphism in the DNA. For example, PCR may be used to amplify anappropriate fragment of genomic DNA which may then be sequenced.Alternatively, the DNA may be subjected to allele-specificoligonucleotide hybridization (in which appropriate oligonucleotides arehybridized to the DNA under conditions permitting detection of a singlebase mismatch) or to a single nucleotide extension assay (in which anoligonucleotide that hybridizes immediately adjacent to the position ofthe polymorphism is extended with one or more labeled nucleotides). Inaddition, traditional restriction fragment length polymorphism analysis(using restriction enzymes that provide differential digestion of thegenomic DNA depending on the presence or absence of the polymorphism)may be performed. Arrays with nucleotide sequences of the presentinvention can be used to detect polymorphisms. The array can comprisemodified nucleotide sequences of the present invention in order todetect the nucleotide sequences of the present invention. In thealternative, any one of the nucleotide sequences of the presentinvention can be placed on the array to detect changes from thosesequences.

[0280] Alternatively a polymorphism resulting in a change in the aminoacid sequence could also be detected by detecting a corresponding changein amino acid sequence of the protein, e.g., by an antibody specific tothe variant sequence.

[0281] 4.7.18 Arthritis and Inflammation

[0282] The immunosuppressive effects of the compositions of theinvention against rheumatoid arthritis is determined in an experimentalanimal model system. The experimental model system is adjuvant inducedarthritis in rats, and the protocol is described by J. Holoshitz, etat., 1983. Science. 219:56, or by B. Waksman et al. 1963. Int. Arch.Allergy Appl. Immunol., 23:129. Induction of the disease can be causedby a single injection, generally intradermally, of a suspension ofkilled Mycobacterium tuberculosis in complete Freund's adjuvant (CFA).The route of injection can vary, but rats may be injected at the base ofthe tail with an adjuvant mixture. The polypeptide is administered inphosphate buffered solution (PBS) at a dose of about 1-5 mg/kg. Thecontrol consists of administering PBS only.

[0283] The procedure for testing the effects of the test compound wouldconsist of intradermally injecting killed Mycobacterium tuberculosis inCFA followed by immediately administering the test compound andsubsequent treatment every other day until day 24. At 14, 15, 18, 20,22, and 24 days after injection of Mycobacterium CFA, an overallarthritis score may be obtained as described by J. Holoskitz above. Ananalysis of the data would reveal that the test compound would have adramatic affect on the swelling of the joints as measured by a decreaseof the arthritis score.

4.8 Therapeutic Methods

[0284] The compositions (including polypeptide fragments, analogs,variants and antibodies or other binding partners or modulatorsincluding antisense polynucleotides) of the invention have numerousapplications in a variety of therapeutic methods. Examples oftherapeutic applications include, but are not limited to, thoseexemplified herein.

[0285] 4.8.1 Example

[0286] One embodiment of the invention is the administration of aneffective amount of the stem cell grouch factor-like polypeptides orother composition of the invention to individuals affected by a diseaseor disorder that can be modulated by regulating the peptides of theinvention. While the mode of administration is not particularlyimportant, parenteral administration is preferred. An exemplary mode ofadministration is to deliver an intravenous bolus. The dosage of stemcell growth factor-like polypeptides or other composition of theinvention will normally be determined by the prescribing physician. Itis to be expected that the dosage will vary according to the age,weight, condition and response of the individual patient. Typically, theamount of polypeptide administered per dose will be in the range ofabout 0.01 μg/kg, to 100 mg/kg of body weight, with the preferred dosebeing about 0.1 μ/kg to 10 mg/kg of patient body weight. For parenteraladministration, stem cell growth factor-like polypeptides of theinvention will be formulated in an injectable form combined with apharmaceutically acceptable parenteral vehicle. Such vehicles are wellknown in the art and examples include water, saline. Ringer's solution,dextrose solution, and solutions consisting of small amounts of thehuman serum albumin. The vehicle may contain minor amounts of additivesthat maintain the isotonicity and stability of the polypeptide or otheractive ingredient. The preparation of such solutions is within the skillof the art.

4.9 Pharmaceutical Formulations and Routes of Administration

[0287] A protein or other composition of the present invention (fromwhatever source derived, including without limitation from recombinantand non-recombinant sources and including antibodies and other bindingpartners of the polypeptides of the invention) may be administered to apatient in need, by itself, or in pharmaceutical compositions where itis mixed with suitable carriers or excipient(s) at doses to treat orameliorate a variety of disorders. Such a composition may optionallycontain (in addition to protein or other active ingredient and acarrier) diluents, fillers, salts, buffers, stabilizers, solubilizers,and other materials well known in the art. The term “pharmaceuticallyacceptable” means a non-toxic material that does not interfere with theeffectiveness of the biological activity of the active ingredient(s).The characteristics of the carrier will depend on the route ofadministration. The pharmaceutical composition of the invention may alsocontain cytokines, lymphokines, or other hematopoietic factors such asM-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IFN, TNF0, TNF1, TNF2,G-CSF, Meg-CSF, thrombopoietin, stem cell factor, and erythropoietin. Infurther compositions, proteins of the invention may be combined withother agents beneficial to the treatment of the disease or disorder inquestion. These agents include various growth factors such as epidermalgrowth factor (EGF), platelet-derived growth factor (PDGF), transforminggrowth factors (TGF-α and TGF-β), insulin-like growth factor (IGF), aswell as cytokines described herein.

[0288] The pharmaceutical composition may further contain other agentswhich either enhance the activity of the protein or other activeingredient or complement its activity or use in treatment. Suchadditional factors and/or agents may be included in the pharmaceuticalcomposition to produce a synergistic effect with protein or other activeingredient of the invention, or to minimize side effects. Conversely,protein or other active ingredient of the present invention may beincluded in formulations of the particular clotting factor, cytokine,lymphokine, other hematopoietic factor, thrombolytic or anti-thromboticfactor, or anti-inflammatory agent to minimize side effects of theclotting factor, cytokine, lymphokine, other hematopoietic factor,thrombolytic or anti-thrombotic factor, or anti-inflammatory agent (suchas IL-1Ra, IL-1 Hy1, IL-1 Hy2, anti-TNF, corticosteroids,immunosuppressive agents). A protein of the present invention may beactive in multimers (e.g., heterodimers or homodimers) or complexes withitself or other proteins. As a result, pharmaceutical compositions ofthe invention may comprise a protein of the invention in such multimericor complexed form.

[0289] As an alternative to being included in a pharmaceuticalcomposition of the invention including a first protein, a second proteinor a therapeutic agent may be concurrently administered with the firstprotein (e.g., at the same time, or at differing times provided thattherapeutic concentrations of the combination of agents is achieved atthe treatment site). Techniques for formulation and administration ofthe compounds of the instant application may be found in “Remington'sPharmaceutical Sciences.” Mack Publishing Co., Easton, Pa., latestedition. A therapeutically effective dose further refers to that amountof the compound sufficient to result in amelioration of symptoms, e g.,treatment, healing, prevention or amelioration of the relevant medicalcondition, or an increase in rate of treatment, healing, prevention oramelioration of such conditions. When applied to an individual activeingredient, administered alone, a therapeutically effective dose refersto that ingredient alone. When applied to a combination, atherapeutically effective dose refers to combined amounts of the activeingredients that result in the therapeutic effect, whether administeredin combination, serially or simultaneously.

[0290] In practicing the method of treatment or use of the presentinvention, a therapeutically effective amount of protein or other activeingredient of the present invention is administered to a mammal having acondition to be treated. Protein or other active ingredient of thepresent invention may be administered in accordance with the method ofthe invention either alone or in combination with other therapies suchas treatments employing cytokines, lymphokines or other hematopoieticfactors. When co-administered with one or more cytokines, lymphokines orother hematopoietic factors, protein or other active ingredient of thepresent invention may be administered either simultaneously with thecytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolyticor anti-thrombotic factors, or sequentially. If administeredsequentially. the attending physician will decide on the appropriatesequence of administering protein or other active ingredient of thepresent invention in combination with cytokine(s), lymphokine(s), otherhematopoietic factor(s), thrombolytic or anti-thrombotic factors.

[0291] 4.9.1 Routes of Administration

[0292] Suitable routes of administration may, for example include oral,rectal, transmucosal, or intestinal administration; parenteral delivery,including intramuscular, subcutaneous, intramedullary injections, aswell as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections. Administrationof protein or other active ingredient of the present invention used inthe pharmaceutical composition or to practice the method of the presentinvention can be carried out in a variety of conventional ways, such asoral ingestion, inhalation, topical application or cutaneous,subcutaneous, intraperitoneal, parenteral or intravenous injection.Intravenous administration to the patient is preferred.

[0293] Alternately, one may administer the compound in a local ratherthan systemic manner, for example, via injection of the compounddirectly into a arthritic joints or in fibrotic tissue, often in a depotor sustained release formulation. In order to prevent the scarringprocess frequently occurring as complication of glaucoma surgery, thecompounds may be administered topically, for example, as eye drops.Furthermore, one may administer the drug in a targeted drug deliverysystem, for example, in a liposome coated with a specific antibody,targeting, for example. arthritic or fibrotic tissue. The liposomes willbe targeted to and taken up selectively by the afflicted tissue.

[0294] The polypeptides of the invention are administered by any routethat delivers an effective dosage to the desired site of action. Thedetermination of a suitable route of administration and an effectivedosage for a particular indication is within the level of skill in theart. Preferably for wound treatment, one administers the therapeuticcompound directly to the site. Suitable dosage ranges for thepolypeptides of the invention can be extrapolated from these dosages orfrom similar studies in appropriate animal models. Dosages can then beadjusted as necessary by the clinician to provide maximal therapeuticbenefit.

[0295] 4.9.2 Compositions/Formulations

[0296] Pharmaceutical compositions for use in accordance with thepresent invention thus may be formulated in a conventional manner usingone or more physiologically acceptable carriers comprising excipientsand auxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. These pharmaceuticalcompositions may be manufactured in a manner that is itself known e.g.,by means of conventional mixing, dissolving, granulating, dragee-making,levigating, emulsifying, encapsulating, entrapping or lyophilizingprocesses. Proper formulation is dependent upon the route ofadministration chosen. When a therapeutically effective amount ofprotein or other active ingredient of the present invention isadministered orally, protein or other active ingredient of the presentinvention will be in the form of a tablet, capsule, powder, solution orelixir. When administered in tablet form, the pharmaceutical compositionof the invention may additionally contain a solid carrier such as agelatin or an adjuvant. The tablet, capsule, and powder contain fromabout 5 to 95% protein or other active ingredient of the presentinvention, and preferably from about 25 to 90% protein or other activeingredient of the present invention. When administered in liquid form, aliquid carrier such as water, petroleum, oils of animal or plant originsuch as peanut oil, mineral oil. soybean oil, or sesame oil, orsynthetic oils may be added. The liquid form of the pharmaceuticalcomposition may further contain physiological saline solution, dextroseor other saccharide solution, or glycols such as ethylene glycol,propylene glycol or polyethylene glycol. When administered in liquidform, the pharmaceutical composition contains from about 0.5 to 90% byweight of protein or other active ingredient of the present invention,and preferably from about 1 to 50% protein or other active ingredient ofthe present invention.

[0297] When a therapeutically effective amount of protein or otheractive ingredient of the present invention is administered byintravenous, cutaneous or subcutaneous injection, protein or otheractive ingredient of the present invention will be in the form of apyrogen-free, parenterally acceptable aqueous solution. The preparationof such parenterally acceptable protein or other active ingredientsolutions, having due regard to pH, isotonicity, stability, and thelike, is within the skill in the art. A preferred pharmaceuticalcomposition for intravenous, cutaneous, or subcutaneous injection shouldcontain, in addition to protein or other active ingredient of thepresent invention, an isotonic vehicle such as Sodium ChlorideInjection, Ringer's Injection, Dextrose Injection, Dextrose and SodiumChloride Injection, Lactated Ringer's Injection, or other vehicle asknown in the art. The pharmaceutical composition of the presentinvention may also contain stabilizers, preservatives, buffers,antioxidants, or other additives known to those of skill in the art. Forinjection, the agents of the invention may be formulated in aqueoussolutions, preferably in physiologically compatible buffers such asHanks's solution, Ringer's solution, or physiological saline buffer. Fortransmucosal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art.

[0298] For oral administration, the compounds can be formulated readilyby combining the active compounds with pharmaceutically acceptablecarriers well known in the art. Such carriers enable the compounds ofthe invention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a patient to be treated. Pharmaceutical preparations fororal use can be obtained solid excipient optionally grinding a resultingmixture, and processing the mixture of granules, after adding suitableauxiliaries, if desired, to obtain tablets or dragee cores. Suitableexcipients are, in particular, fillers such as sugars, includinglactose, sucrose, mannitol, or sorbitol; cellulose preparations such as,for example, maize starch, wheat starch, rice starch, potato starch,gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate. Dragee cores areprovided with suitable coatings. For this purpose, concentrated sugarsolutions may be used, which may optionally contain gum arabic, talc,polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments may be added to the tablets ordragee coatings for identification or to characterize differentcombinations of active compound doses.

[0299] Pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft sealed capsules madeof gelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for suchadministration. For buccal administration, the compositions may take theform of tablets or lozenges formulated in conventional manner.

[0300] For administration by inhalation the compounds for use accordingto the present invention are conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebuliser withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g., gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch. The compounds maybe formulated for parenteral administration by injection, e.g., by bolusinjection or continuous infusion. Formulations for injection may bepresented in unit dosage form e.g., in ampules or in multi-dosecontainers with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory, agents such as suspending,stabilizing and/or dispersing agents.

[0301] Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

[0302] The compounds may also be formulated in rectal compositions suchas suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides. In additionto the formulations described previously, the compounds may also beformulated as a depot preparation. Such long acting formulations may beadministered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

[0303] A pharmaceutical carrier for the hydrophobic compounds of theinvention is a co-solvent system comprising benzyl alcohol, a nonpolarsurfactant, a water-miscible organic polymer, and an aqueous phase. Theco-solvent system may be the VPD co-solvent system. VPD is a solution of3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80,and 65% w/v polyethylene glycol 300, made up to volume in absoluteethanol. The VPD co-solvent system (VPD:5W) consists of VPD diluted 1:1with a 5% dextrose in water solution. This co-solvent system dissolveshydrophobic compounds well and itself produces low toxicity uponsystemic administration. Naturally, the proportions of a co-solventsystem may be varied considerably without destroying its solubility andtoxicity characteristics. Furthermore, the identity of the co-solventcomponents may be varied: for example, other low-toxicity nonpolarsurfactants may be used instead of polysorbate 80; the fraction size ofpolyethylene glycol may be varied; other biocompatible polymers mayreplace polyethylene glycol, e.g. polyvinyl pyrrolidone; and othersugars or polysaccharides may substitute for dextrose. Alternatively,other delivery systems for hydrophobic pharmaceutical compounds may beemployed. Liposomes and emulsions are well known examples of deliveryvehicles or carriers for hydrophobic drugs. Certain organic solventssuch as dimethylsulfoxide also may be employed, although usually at thecost of reater toxicity. Additionally, the compounds may be deliveredusing a sustained-release system, such as semipermeable matrices ofsolid hydrophobic polymers containing the therapeutic agent. Varioustypes of sustained-release materials have been established and are wellknown by those skilled in the art. Sustained-release capsules may,depending on their chemical nature, release the compounds for a fewweeks up to over 100 days. Depending on the chemical nature and thebiological stability of the therapeutic reagent, additional strategiesfor protein or other active ingredient stabilization may be employed.

[0304] The pharmaceutical compositions also may comprise suitable solidor gel phase carriers or excipients. Examples of such carriers orexcipients include but are not limited to calcium carbonate, calciumphosphate, various sugars, starches, cellulose derivatives, gelatin, andpolymers such as polyethylene glycols. Many of the active ingredients ofthe invention may be provided as salts with pharmaceutically compatiblecounter ions. Such pharmaceutically acceptable base addition salts arethose salts which retain the biological effectiveness and properties ofthe free acids and which are obtained by reaction with inorganic ororganic bases such as sodium hydroxide, magnesium hydroxide, ammonia,trialkylamine, dialkylamine, monoalkylamine, dibasic amino acids, sodiumacetate, potassium benzoate, triethanol amine and the like.

[0305] The pharmaceutical composition of the invention may be in theform of a complex of the protein(s) or other active ingredient ofpresent invention along with protein or peptide antigens. The proteinand/or peptide antigen will deliver a stimulatory signal to both B and Tlymphocytes. B lymphocytes will respond to antigen through their surfaceimmunoglobulin receptor. T lymphocytes will respond to antigen throughthe T cell receptor (TCR) following presentation of the antigen by MHCproteins. MHC and structurally related proteins including those encodedby class I and class II MHC genes on host cells will serve to presentthe peptide antigen(s) to T lymphocytes. The antigen components couldalso be supplied as purified MHC-peptide complexes alone or withco-stimulatory molecules that can directly signal T cells. Alternativelyantibodies able to bind surface immunoglobulin and other molecules on Bcells as well as antibodies able to bind the TCR and other molecules onT cells can be combined with the pharmaceutical composition of theinvention.

[0306] The pharmaceutical composition of the invention may be in theform of a liposome in which protein of the present invention iscombined, in addition to other pharmaceutically acceptable carriers,with amphipathic agents such as lipids which exist in aggregated form asmicelles, insoluble monolayers, liquid crystals, or lamellar layers inaqueous solution. Suitable lipids for liposomal formulation include,without limitation, monoglycerides, diglycerides. sulfatides,lysolecithins, phospholipids, saponin, bile acids, and the like.Preparation of such liposomal formulations is within the level of skillin the art, as disclosed, for example, in U.S. Pat. Nos. 4,235,871;4,501,728; 4,837,028; and 4,737,323, all of which are incorporatedherein by reference.

[0307] The amount of protein or other active ingredient of the presentinvention in the pharmaceutical composition of the present inventionwill depend upon the nature and severity of the condition being treated,and on the nature of prior treatments which the patient has undergone.Ultimately, the attending physician will decide the amount of protein orother active ingredient of the present invention with which to treateach individual patient. Initially, the attending physician willadminister low doses of protein or other active ingredient of thepresent invention and observe the patient's response. Larger doses ofprotein or other active ingredient of the present invention may beadministered until the optimal therapeutic effect is obtained for thepatient, and at that point the dosage is not increased further. It iscontemplated that the various pharmaceutical compositions used topractice the method of the present invention should contain about 0.01μg to about 100 mg (preferably about 0.1 μg to about 10 mg, morepreferably about 0.1 μg to about 1 mg) of protein or other activeingredient of the present invention per kg, body weight. Forcompositions of the present invention which are useful for bone,cartilage, tendon or ligament regeneration, the therapeutic methodincludes administering the composition topically, systematically, orlocally as an implant or device. When administered, the therapeuticcomposition for use in this invention is, of course, in a pyrogen-free,physiologically acceptable form. Further, the composition may desirablebe encapsulated or injected in a viscous form for delivery to the siteof bone, cartilage or tissue damage. Topical administration may besuitable for wound healing and tissue repair. Therapeutically usefulagents other than a protein or other active ingredient of the inventionwhich may also optionally be included in the composition as describedabove, may alternatively or additionally, be administered simultaneouslyor sequentially with the composition in the methods of the invention.Preferably for bone and/or cartilage formation, the composition wouldinclude a matrix capable of delivering the protein-containing or otheractive ingredient-containing composition to the site of bone and/orcartilage damage, providing a structure for the developing bone andcartilage and optimally capable of being resorbed into the body. Suchmatrices may be formed of materials presently in use for other implantedmedical applications.

[0308] The choice of matrix material is based on biocompatibility,biodegradability, mechanical properties, cosmetic appearance andinterface properties. The particular application of the compositionswill define the appropriate formulation. Potential matrices for thecompositions may be biodegradable and chemically defined calciumsulfate, tricalcium phosphate, hydroxyapatite, polylactic acid,polyglycolic acid and polyanhydrides. Other potential materials arebiodegradable and biologically well-defined, such as bone or dermalcollagen. Further matrices are comprised of pure proteins orextracellular matrix components. Other potential matrices arenonbiodegradable and chemically defined, such as sinteredhydroxyapatite, bioglass, aluminates, or other ceramics. Matrices may becomprised of combinations of any of the above mentioned types ofmaterial, such as polylactic acid and hydroxyapatite or collagen andtricalcium phosphate. The bioceramics may be altered in composition,such as in calcium-aluminate-phosphate and processing to alter poresize, particle size, particle shape, and biodegradability. Presentlypreferred is a 50:50 (mole weight) copolymer of lactic acid and glycolicacid in the form of porous particles having diameters ranging from 150to 800 microns. In some applications, it will be useful to utilize asequestering agent, such as carboxymethyl cellulose or autologous bloodclot, to prevent the protein compositions from disassociating from thematrix.

[0309] A preferred family of sequestering agents is cellulosic materialssuch as alkylcelluloses (including hydroxyalkylcelluloses), includingmethylcellulose, ethylcellulose. hydroxyethylcellulose,hydroxypropylcellulose, hydroxypropyl-methylcellulose, andcarboxymethylcellulose, the most preferred being cationic salts ofcarboxymethylcellulose (CMC). Other preferred sequestering agentsinclude hyaluronic acid, sodium alginate. poly(ethylene glycol),polyoxyethylene oxide, carboxyvinyl polymer and poly(vinyl alcohol). Theamount of sequestering agent useful herein is 0.5-20 wt %, preferably1-10 wt % based on total formulation weight, which represents the amountnecessary to prevent desorption of the protein from the polymer matrixand to provide appropriate handling of the composition, yet not so muchthat the progenitor cells are prevented from infiltrating the matrix,thereby providing the protein the opportunity to assist the osteogenicactivity of the progenitor cells. In further compositions, proteins orother active ingredient of the invention may be combined with otheragents beneficial to the treatment of the bone and/or cartilage defect,wound, or tissue in question. These agents include various growthfactors such as epidermal growth factor (EGF). platelet derived growthfactor (PDGF), transforming growth factors (TGF-α and TGF-β), andinsulin-like growth factor (IGF).

[0310] The therapeutic compositions are also presently valuable forveterinary applications. Particularly domestic animals and thoroughbredhorses, in addition to humans, are desired patients for such treatmentwith proteins or other active ingredient of the present invention. Thedosage regimen of a protein-containing pharmaceutical composition to beused in tissue regeneration will be determined by the attendingphysician considering various factors which modify the action of theproteins, e.g., amount of tissue weight desired to be formed, the siteof damage, the condition of the damaged tissue, the size of a wound,type of damaged tissue (e.g., bone), the patient's age, sex, and diet,the severity of any infection, time of administration and other clinicalfactors. The dosage may vary with the type of matrix used in thereconstitution and with inclusion of other proteins in thepharmaceutical composition. For example, the addition of other knowngrowth factors, such as IGF I (insulin like growth factor I), to thefinal composition, may also effect the dosage. Progress can be monitoredby periodic assessment of tissue/bone growth and/or repair, for example,X-rays, histomorphometric determinations and tetracycline labeling.

[0311] Polynucleotides of the present invention can also be used forgene therapy. Such polynucleotides can be introduced either in vivo orex vivo into cells for expression in a mammalian subject.Polynucleotides of the invention may also be administered by other knownmethods for introduction of nucleic acid into a cell or organism(including, without limitation, in the form of viral vectors or nakedDNA). Cells may also be cultured ex vivo in the presence of proteins ofthe present invention in order to proliferate or to produce a desiredeffect on or activity in such cells. Treated cells can then beintroduced in vivo for therapeutic purposes.

[0312] 4.9.3 Effective Dosage

[0313] Pharmaceutical compositions suitable for use in the presentinvention include compositions wherein the active ingredients arecontained in an effective amount to achieve its intended purpose. Morespecifically, a therapeutically effective amount means an amounteffective to prevent development of or to alleviate the existingsymptoms of the subject being treated. Determination of the effectiveamount is well within the capability of those skilled in the art,especially in light of the detailed disclosure provided herein. For anycompound used in the method of the invention, the therapeuticallyeffective dose can be estimated initially from appropriate in vitroassays. For example, a dose can be formulated in animal models toachieve a circulating concentration range that can be used to moreaccurately determine useful doses in humans. For example, a dose can beformulated in animal models to achieve a circulating concentration rangethat includes the IC₅₀ as determined in cell culture (i.e., theconcentration of the test compound which achieves a half-maximalinhibition of the protein's biological activity). Such information canbe used to more accurately determine useful doses in humans.

[0314] A therapeutically effective dose refers to that amount of thecompound that results in amelioration of symptoms or a prolongation ofsurvival in a patient. Toxicity and therapeutic efficacy of suchcompounds can be determined by standard pharmaceutical procedures incell cultures or experimental animals, e.g. for determining the LD₅₀(the dose lethal to 50% of the population) and the ED₅₀ (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index and itcan be expressed as the ratio between LD₅₀ and ED₅₀. Compounds whichexhibit high therapeutic indices are preferred. The data obtained fromthese cell culture assays and animal studies can be used in formulatinga range of dosage for use in human. The dosage of such compounds liespreferably within a range of circulating concentrations that include theED₅₀ with little or no toxicity. The dosage may vary within this rangedepending upon the dosage form employ ed and the route of administrationutilized. The exact formulation, route of administration and dosage canbe chosen by the individual physician in view of the patient'scondition. See, e.g., Fingl et al., 1975, in “The Pharmacological Basisof Therapeutics”. Ch. 1 p. 1. Dosage amount and interval may be adjustedindividually to provide plasma levels of the active moiety which aresufficient to maintain the desired effects, or minimal effectiveconcentration (MEC). The MEC will vary for each compound but can beestimated from in vitro data. Dosages necessary to achieve the MEC willdepend on individual characteristics and route of administration.However, HPLC assays or bioassays can be used to determine plasmaconcentrations.

[0315] Dosage intervals can also be determined using MEC value.Compounds should be administered using a regimen which maintains plasmalevels above the MEC for 10-90% of the time, preferably between 30-90%and most preferably between 50-90%. In cases of local administration orselective uptake, the effective local concentration of the drug may notbe related to plasma concentration.

[0316] An exemplary dosage regimen for polypeptides or othercompositions of the invention will be in the range of about 0.01 μg/kgto 100 mg/kg of body weight daily, with the preferred dose being about0.1 μg/kg to 25 mg/kg of patient body weight daily, varying in adultsand children. Dosing may be once daily, or equivalent doses may bedelivered at longer or shorter intervals.

[0317] The amount of composition administered will, of course, bedependent on the subject being treated, on the subject's age and weight,the severity of the affliction, the manner of administration and thejudgment of the prescribing physician.

[0318] 4.9.4 Packaging

[0319] The compositions may, if desired, be presented in a pack ordispenser device which may contain one or more unit dosage formscontaining the active ingredient. The pack may, for example, comprisemetal or plastic foil, such as a blister pack. The pack or dispenserdevice may be accompanied by instructions for administration.Compositions comprising a compound of the invention formulated in acompatible pharmaceutical carrier may also be prepared, placed in anappropriate container, and labeled for treatment of an indicatedcondition.

4.10 Antibodies

[0320] 4.10.1 Human Antibodies

[0321] Fully human antibodies relate to antibody molecules in whichessentially the entire sequences of both the light chain and the heavychain, including the CDRs, arise from human genes. Such antibodies aretermed “human antibodies”, or “fully human antibodies” herein. Humanmonoclonal antibodies can be prepared by the trioma technique; the humanB-cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4:72) and the EBV hybridoma technique to produce human monoclonalantibodies (see Cole et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCERTHERAPY. Alan R. Liss. Inc., pp. 77-96). Human monoclonal antibodies maybe utilized in the practice of the present invention and may be producedby using human hybridomas (see Cote, et al., 1983. Proc Natl Acad SciUSA 80: 2026-2030) or by transforming human B-cells with Epstein BarrVirus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES ANDCANCER THERAPY. Alan R. Liss. Inc., pp. 77-96). In addition, humanantibodies can also be produced using additional techniques, includingphage display libraries (Hoogenboom and Winter. J. Mol. Biol., 227:381(1991): Marks et al., J. Mol. Biol., 222:581 (1991)). Similarly, humanantibodies can be made by introducing human immunoglobulin loci intotransgenic animals, e.g., mice in which the endogenous immunoglobulingenes have been partially or completely inactivated. Upon challenge,human antibody production is observed, which closely resembles that seenin humans in all respects, including gene rearrangement, assembly, andantibody repertoire. This approach is described, for example, in U.S.Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425;5,661,016, and in Marks et al, (Bio/Technology 10, 779-783 (1992));Lonberg et al, (Nature 368 856-859 (1994)); Morrison (Nature 368, 812-13(1994)); Fishwild et al, (Nature Biotechnology 14, 845-51 (1996));Neuberger (Nature Biotechnology 14, 826 (1996)); and Lonberg and Huszar(Intern. Rev. Immunol. 13 65-93 (1995)).

[0322] Human antibodies may additionally be produced using transgenicnonhuman animals which are modified so as to produce fully humanantibodies rather than the animal's endogenous antibodies in response tochallenge by an antigen. (See PCT publication WO94/02602). Theendogenous genes encoding the heavy and light immunoglobulin chains inthe nonhuman host have been incapacitated, and active loci encodinghuman heavy and light chain immunoglobulins are inserted into the host'sgenome. The human genes are incorporated, for example, using yeastartificial chromosomes containing the requisite human DNA segments. Ananimal which provides all the desired modifications is then obtained asprogeny by crossbreeding intermediate transgenic animals containingfewer than the full complement of the modifications. The preferredembodiment of such a nonhuman animal is a mouse, and is termed theXenomouse™ as disclosed in PCT publications WO 96/33735 and WO 96/34096.This animal produces B cells which secrete fully human immunoglobulins.The antibodies can be obtained directly from the animal afterimmunization with an immunogen of interest, as, for example, apreparation of a polyclonal antibody, or alternatively from immortalizedB cells derived from the animal, such as hybridomas producing monoclonalantibodies. Additionally, the genes encoding the immunoglobulins withhuman variable regions can be recovered and expressed to obtain theantibodies directly, or can be further modified to obtain analogs ofantibodies such as, for example, single chain Fv molecules.

[0323] An example of a method of producing a nonhuman host, exemplifiedas a mouse, lacking expression of an endogenous immunoglobulin heavychain is disclosed in U.S. Pat. No. 5,939,598. It can be obtained by amethod including deleting the J segment genes from at least oneendogenous heavy chain locus in an embryonic stem cell to preventrearrangement of the locus and to prevent formation of a transcript of arearranged immunoglobulin heavy chain locus, the deletion being effectedby a targeting vector containing a gene encoding a selectable marker;and producing from the embryonic stem cell a transgenic mouse whosesomatic and germ cells contain the gene encoding the selectable marker.

[0324] A method for producing an antibody of interest, such as a humanantibody, is disclosed in U.S. Pat. No. 5,916,771. It includesintroducing an expression vector that contains a nucleotide sequenceencoding a heavy chain into one mammalian host cell in culture,introducing an expression vector containing a nucleotide sequenceencoding a light chain into another mammalian host cell, and fusing thetwo cells to form a hybrid cell. The hybrid cell expresses an antibodycontaining the heavy chain and the light chain.

[0325] In a further improvement on this procedure, a method foridentifying a clinically relevant epitope on an immunogen, and acorrelative method for selecting an antibody that bindsimmunospecifically to the relevant epitope with high affinity, aredisclosed in PCT publication WO 99/53049.

[0326] 4.10.2 Fab Fragments and Single Chain Antibodies

[0327] According to the invention, techniques can be adapted for theproduction of single-chain antibodies specific to an antigenic proteinof the invention (see e.g., U.S. Pat. No. 4,946,778). In addition,methods can be adapted for the construction of F_(ab) expressionlibraries (see e.g., Huse, et al., 1989 Science 246: 1275-1281 ) toallow rapid and effective identification of monoclonal F_(ab) fragmentswith the desired specificity for a protein or derivatives, fragments,analogs or homologs thereof. Antibody fragments that contain theidiotypes to a protein antigen may be produced by techniques known inthe art including, but not limited to: (i) an F_((ab′)2) fragmentproduced by pepsin digestion of an antibody molecule; (ii) an F_(ab)fragment generated by reducing the disulfide bridges of an F_((ab′)2)fragment; (iii) an F_(ab) fragment generated by the treatment of theantibody molecule with papain and a reducing agent and (iv) F_(v)fragments.

[0328] 4.10.3 Bispecific Antibodies

[0329] Bispecific antibodies are monoclonal, preferably human orhumanized, antibodies that have binding specificities for at least twodifferent antigens. In the present case, one of the bindingspecificities is for an antigenic protein of the invention. The secondbinding target is any other antigen, and advantageously is acell-surface protein or receptor or receptor subunit.

[0330] Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, Nature, 305:537-539 (1983)). Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of ten different antibodymolecules, of which only one has the correct bispecific structure. Thepurification of the correct molecule is usually accomplished by affinitychromatography steps. Similar procedures are disclosed in WO 93/08829,published 13 May 1993, and in Traunecker et al., 1991 EMBO J.,10:3655-3659.

[0331] Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Forfurther details of generating bispecific antibodies see, for example.Suresh et al., Methods in Enzymology, 121:210 (1986).

[0332] According to another approach described in WO 96/27011, theinterface between a pair of antibody molecules can be engineered tomaximize the percentage of heterodimers which are recovered fromrecombinant cell culture. The preferred interface comprises at least apart of the CH3 region of an antibody constant domain. In this method,one or more small amino acid side chains from the interface of the firstantibody molecule are replaced with larger side chains (e.g. tyrosine ortryptophan). Compensatory “cavities” of identical or similar size to thelarge side chain(s) are created on the interface of the second antibodymolecule by replacing large amino acid side chains with smaller ones(e.g, alanine or threonine). This provides a mechanism for increasingthe yield of the heterodimer over other unwanted end-products such ashomodimers.

[0333] Bispecific antibodies can be prepared as full-length antibodiesor antibody fragments (e.g. F(ab′)₂ bispecific antibodies). Techniquesfor generating bispecific antibodies from antibody fragments have beendescribed in the literature. For example, bispecific antibodies can beprepared using chemical linkage. Brennan et al., Science 229:81 (1985)describe a procedure wherein intact antibodies are proteolyticallycleaved to generate F(ab′)₂ fragments. These fragments are reduced inthe presence of the dithiol complexing agent sodium arsenite tostabilize vicinal dithiols and prevent intermolecular disulfideformation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

[0334] Additionally. Fab′ fragments can be directly recovered from E.coli and chemically coupled to form bispecific antibodies. Shalaby etal., J. Exp. Med. 175:217-225 (1992) describe the production of a fullyhumanized bispecific antibody F(ab′)₂ molecule. Each Fab′ fragment wasseparately secreted from E. coli and subjected to directed chemicalcoupling in vitro to form the bispecific antibody. The bispecificantibody thus formed was able to bind to cells overexpressing the ErbB2receptor and normal human T cells, as well as trigger the lytic activityof human cytotoxic lymphokines against human breast tumor targets.

[0335] Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol. 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See. Gruber et al., J. Immunol. 152:5368 (1994).

[0336] Antibodies with more than two valencies are contemplated. Forexample, trispecific antibodies can be prepared. Tutt et al., J.Immunol. 147:60 (1991).

[0337] Exemplary bispecific antibodies can bind to two differentepitopes, at least one of which originates in the protein antigen of theinvention. Alternatively, an anti-antigenic arm of an immunoglobulinmolecule can be combined with an arm which binds to a triggeringmolecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2,CD3, CD28, or B7), or Fc receptors for IgG (Fc R), such as Fe RI (CD64),Fc RII (CD32) and Fc RIII (CD16) so as to focus cellular defensemechanisms to the cell expressing the particular antigen. Bispecificantibodies can also be used to direct cytotoxic agents to cells whichexpress a particular antigen. These antibodies possess anantigen-binding arm and an arm which binds a cytotoxic agent or aradionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. Anotherbispecific antibody of interest binds the protein antigen describedherein and further binds tissue factor (TF).

[0338] 4.10.4 Heteroconjugate Antibodies

[0339] Heteroconjugate antibodies are also within the scope of thepresent invention. Heteroconjugate antibodies are composed of twocovalently joined antibodies. Such antibodies have, for example, beenproposed to target immune system cells to unwanted cells (U.S. Pat. No.4,676,980), and for treatment of HIV infection (WO 91/00360: WO92/200373; EP 03089). It is contemplated that the antibodies can beprepared in vitro using knows methods in synthetic protein chemistry,including those involving crosslinking agents. For example, immunotoxinscan be constructed using a disulfide exchange reaction or by, forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-1-4-mercaptobutyrimidate and those disclosed,for example, in U.S. Pat. No. 4,676,980.

[0340] 4.10.5 Effector Function Engineering

[0341] It can be desirable to modify the antibody of the invention withrespect to effector function, so as to enhance, e.g., the effectivenessof the antibody in treating cancer. For example, cysteine residue(s) canbe introduced into the Fc region, thereby allowing interchain disulfidebond formation in this region. The homodimeric antibody thus generatedcan have improved internalization capability and/or increasedcomplement-mediated cell killing and antibody-dependent cellularcytotoxicity (ADCC). See Caron et al., J. Exp Med., 176: 1191-1195(1992) and Shopes. J. Immunol., 148: 2918-2922 (1992). Homodimericantibodies with enhanced anti-tumor activity can also be prepared usingheterobifunctional cross-linkers as described in Wolff et al, CancerResearch, 53: 2560-2565 (1993). Alternatively, an antibody can beengineered that has dual Fc regions and can thereby have enhancedcomplement lysis and ADCC capabilities. See Stevenson et al.,Anti-Cancer Drug Design. 3: 219-230(1989).

[0342] 4.10.6 Immunoconjugates

[0343] The invention also pertains to Immunoconjugates comprising anantibody conjugated to a cytotoxic agent such as a chemotherapeuticagent, toxin (e.g., an enzymatically active toxin of bacterial, fungal,plant, or animal origin, or fragments thereof), or a radioactive isotope(i.e., a radioconjugate).

[0344] Chemotherapeutic agents useful in the generation of suchImmunoconjugates have been described above. Enzymatically active toxinsand fragments thereof that can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins. Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitosellin, restrictocin, phenomycin, enomycin, and the tricothecenes. Avariety of radionuclides are available for the production ofradioconjugated antibodies. Examples include ₂₁₂Bi, ₁₃₁In, ₉₀Y, and₈₆Re.

[0345] Conjugates of the antibody and cytotoxic agent are made using avariety of bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP). iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate). and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science, 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

[0346] In another embodiment, the antibody can be conjugated to a“receptor” (such streptavidin) for utilization in tumor pretargetingwherein the antibody-receptor conjugate is administered to the patient,followed by removal of unbound conjugate from the circulation using aclearing agent and then administration of a “ligand” (e.g., avidin) thatis in turn conjugated to a cytotoxic agent.

[0347] 4.11 Computer Readable Sequences

[0348] In one application of this embodiment, a nucleotide sequence ofthe present invention can be recorded on computer readable media. Asused herein, “computer readable media” refers to any medium which can beread and accessed directly by a computer. Such media include, but arenot limited to: magnetic storage media, such as floppy discs, hard discstorage medium, and magnetic tape; optical storage media such as CD-ROM;electrical storage media such as RAM and ROM; and hybrids of thesecategories such as magnetic/optical storage media. A skilled artisan canreadily appreciate how any of the presently known computer readablemediums can be used to create a manufacture comprising computer readablemedium having recorded thereon a nucleotide sequence of the presentinvention. As used herein, “recorded” refers to a process for storinginformation on computer readable medium. A skilled artisan can readilyadopt any of the presently known methods for recording information oncomputer readable medium to generate manufactures comprising thenucleotide sequence information of the present invention.

[0349] A variety of data storage structures are available to a skilledartisan for creating a computer readable medium having recorded thereona nucleotide sequence of the present invention. The choice of the datastorage structure will generally be based on the means chosen to accessthe stored information. In addition, a variety of data processorprograms and formats can be used to store the nucleotide sequenceinformation of the present invention on computer readable medium. Thesequence information can be represented in a word processing text file,formatted in commercially-available software such as WordPerfect andMicrosoft Word, or represented in the form of an ASCII file, stored in adatabase application, such as DB2, Sbase, Oracle, or the like. A skilledartisan can readily adapt any number of data processor structuringformats (e.g, text file or database) in order to obtain computerreadable medium having recorded thereon the nucleotide sequenceinformation of the present invention.

[0350] By providing any of the nucleotide sequences SEQ ID NO: 1-22, 24,26-27, 29, or 33 or a representative fragment thereof; or a nucleotidesequence at least 95% identical to any of the nucleotide sequences ofSEQ ID NO: 1-22, 24, 26-27, 29, or 33 in computer readable form, askilled artisan can routinely access the sequence information for avariety of purposes. Computer software is publicly available whichallows a skilled artisan to access sequence information provided in acomputer readable medium. The examples which follow demonstrate howsoftware which implements the BLAST (Altschul et al., J. Mol. Biol.215:403-410 (1990)) and BLAZE (Brutlag et al., Comp. Chem. 17:203-207(1993)) search algorithms on a Sybase system is used to identify openreading frames (ORFs) within a nucleic acid sequence. Such ORFs may beprotein encoding fragments and may be useful in producing commerciallyimportant proteins such as enzymes used in fermentation reactions and inthe production of commercially useful metabolites.

[0351] As used herein, “a computer-based system” refers to the hardwaremeans, software means, and data storage means used to analyze thenucleotide sequence information of the present invention. The minimumhardware means of the computer-based systems of the present inventioncomprises a central processing unit (CPU), input means, output means,and data storage means. A skilled artisan can readily appreciate thatany one of the currently available computer-based systems are suitablefor use in the present invention. As stated above, the computer-basedsystems of the present invention comprise a data storage means havingstored therein a nucleotide sequence of the present invention and thenecessary hardware means and software means for supporting andimplementing a search means. As used herein. “data storage means” refersto memory which can store nucleotide sequence information of the presentinvention, or a memory access means which can access manufactures havingrecorded thereon the nucleotide sequence information of the presentinvention.

[0352] As used herein, “search means” refers to one or more programswhich are implemented on the computer-based system to compare a targetsequence or target structural motif with the sequence information storedwithin the data storage means. Search means are used to identifyfragments or regions of a known sequence which match a particular targetsequence or target motif. A variety of known algorithms are disclosedpublicly and a variety of commercially available software for conductingsearch means are and can be used in the computer-based systems of thepresent invention. Examples of such software includes, but is notlimited to, Smith-Waterman, MacPattern (EMBL), BLASTN and BLASTA(NPOLYPEPTIDEIA). A skilled artisan can readily recognize that any oneof the available algorithms or implementing software packages forconducting homology searches can be adapted for use in the presentcomputer-based systems. As used herein, a “target sequence” can be anynucleic acid or amino acid sequence of six or more nucleotides or two ormore amino acids. A skilled artisan can readily recognize that thelonger a target sequence is, the less likely a target sequence will bepresent as a random occurrence in the database. The most preferredsequence length of a target sequence is from about 10 to 100 aminoacids, or from about 30 to 300 nucleotide residues. However, it is wellrecognized that searches for commercially important fragments, such assequence fragments involved in gene expression and protein processing,may be of shorter length.

[0353] As used herein, “a target structural motif,” or “target motif,”refers to any, rationally selected sequence or combination of sequencesin which the sequence(s) are chosen based on a three-dimensionalconfiguration which is formed upon the folding of the target motif.There are a variety of target motifs known in the art. Protein targetmotifs include, but are not limited to, enzyme active sites and signalsequences. Nucleic acid target motifs include, but are not limited to,promoter sequences, hairpin structures and inducible expression elements(protein binding sequences).

[0354] 4.12 Triple Helix Formation

[0355] In addition, the fragments of the present invention, as broadlydescribed, can be used to control gene expression through triple helixformation or antisense DNA or RNA, both of which methods are based onthe binding of a polynucleotide sequence to DNA or RNA. Polynucleotidessuitable for use in these methods are usually 20 to 40 bases in lengthand are designed to be complementary to a region of the gene involved intranscription (triple helix—see Lee et al., Nucl. Acids Res. 6:3073(1979); Cooney et al., Science 15241:456 (1988); and Dervan et al.,Science 251:1360 (1991)) or to the mRNA itself (antisense—Olmno. J.Neurochem. 56:560 (1991): Oligodeoxynucleotides as Antisense Inhibitorsof Gene Expression, CRC Press, Boca Raton. Fla. (1988)). Triplehelix-formation optimally results in a shut-off of RNA transcriptionfrom DNA, while antisense RNA hybridization blocks translation of anmRNA molecule into polypeptide. Both techniques have been demonstratedto be effective in model systems. Information contained in the sequencesof the present invention is necessary for the design of an antisense ortriple helix oligonucleotide.

[0356] 4.13 Diagnostic Assays and Kits

[0357] The present invention further provides methods to identify thepresence or expression of one of the ORFs of the present invention, orhomolog thereof, in a test sample, using a nucleic acid probe orantibodies of the present invention, optionally conjugated or otherwiseassociated with a suitable label.

[0358] In general, methods for detecting a polynucleotide of theinvention can comprise contacting a sample with a compound that binds toand forms a complex with the polynucleotide for a period sufficient toform the complex, and detecting the complex, so that if a complex isdetected, a polynucleotide of the invention is detected in the sample.Such methods can also comprise contacting a sample under stringenthybridization conditions with nucleic acid primers that anneal to apolynucleotide of the invention under such conditions, and amplifyingannealed polynucleotides, so that if a polynucleotide is amplified, apolynucleotide of the invention is detected in the sample.

[0359] In general, methods for detecting a polypeptide of the inventioncan comprise contacting a sample with a compound that binds to and formsa complex with the polypeptide for a period sufficient to form thecomplex, and detecting the complex, so that if a complex is detected, apolypeptide of the invention is detected in the sample.

[0360] In detail, such methods comprise incubating a test sample withone or more of the antibodies or one or more of the nucleic acid probesof the present invention and assaying for binding of the nucleic acidprobes or antibodies to components within the test sample.

[0361] Conditions for incubating a nucleic acid probe or antibody with atest sample vary. Incubation conditions depend on the format employed inthe assay, the detection methods employed, and the type and nature ofthe nucleic acid probe or antibody used in the assay. One skilled in theart will recognize that any one of the commonly available hybridization.amplification or immunological assay formats can readily be adapted toemploy the nucleic acid probes or antibodies of the present invention.Examples of such assays can be found in Chard. T., An Introduction toRadioimmunoassay and Related Techniques, Elsevier Science Publishers,Amsterdam, The Netherlands (1986); Bullock. G. R. et al., Techniques inImmunocytochemistry, Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 7(1983), Vol. 3 (1985); Tijssen. P., Practice and Theory of immunoassays:Laboratory Techniques in Biochemistry and Molecular Biology, ElsevierScience Publishers, Amsterdam, The Netherlands (1985). The test samplesof the present invention include cells, protein or membrane extracts ofcells, or biological fluids such as sputum, blood, serum, plasma, orurine. The test sample used in the above-described method will varybased on the assay format, nature of the detection method and thetissues, cells or extracts used as the sample to be assayed. Methods forpreparing protein extracts or membrane extracts of cells are well knownin the art and can be readily be adapted in order to obtain a samplewhich is compatible with the system utilized.

[0362] In another embodiment of the present invention, kits are providedwhich contain the necessary reagents to carry out the assays of thepresent invention. Specifically, the invention provides a compartmentkit to receive, in close confinement, one or more containers whichcomprises: (a) a first container comprising one of the probes orantibodies of the present invention; and (b) one or more othercontainers comprising one or more of the following: wash reagents,reagents capable of detecting presence of a bound probe or antibody.

[0363] In detail, a compartment kit includes any kit in which reagentsare contained in separate containers. Such containers include smallglass containers, plastic containers or strips of plastic or paper. Suchcontainers allows one to efficiently transfer reagents from onecompartment to another compartment such that the samples and reagentsare not cross-contaminated, and the agents or solutions of eachcontainer can be added in a quantitative fashion from one compartment toanother. Such containers will include a container which will accept thetest sample, a container which contains the antibodies used in theassay, containers which contain wash reagents (such as phosphatebuffered saline. Tris-buffers, etc.), and containers which contain thereagents used to detect the bound antibody or probe. Types of detectionreagents include labeled nucleic acid probes, labeled secondaryantibodies, or in the alternative, if the primary antibody is labeled,the enzymatic, or antibody binding reagents which are capable ofreacting with the labeled antibody. One skilled in the art will readilyrecognize that the disclosed probes and antibodies of the presentinvention can be readily incorporated into one of the established kitformats which are well known in the art.

4.14 Medical Imaging

[0364] The novel polypeptides and binding partners of the invention areuseful in medical imaging of sites expressing the molecules of theinvention (e.g., where the polypeptide of the invention is involved inthe immune response, for imaging sites of inflammation or infection).See, e.g., Kunkel et al., U.S. Pat. No. 5,413,778. Such methods involvechemical attachment of a labeling or imaging agent, administration ofthe labeled polypeptide to a subject in a pharmaceutically acceptablecarrier, and imaging the labeled polypeptide in vivo at the target site.

4.15 Screening Assays

[0365] Using the isolated proteins and polynucleotides of the invention,the present invention further provides methods of obtaining andidentifying agents which bind to a polypeptide encoded by an ORFcorresponding to any of the nucleotide sequences set forth in SEQ ID NO:1-22, 24, 26-27, 29, or 33, or bind to a specific domain of thepolypeptide encoded by the nucleic acid. In detail, said methodcomprises the steps of:

[0366] (a) contacting an agent with an isolated protein encoded by anORF of the present invention, or nucleic acid of the invention; and

[0367] (b) determining whether the agent binds to said protein or saidnucleic acid.

[0368] In general, therefore, such methods for identifying compoundsthat bind to a polynucleotide of the invention can comprise contacting acompound with a polynucleotide of the invention for a time sufficient toform a polynucleotide/compound complex, and detecting the complex, sothat if a polynucleotide/compound complex is detected, a compound thatbinds to a polynucleotide of the invention is identified.

[0369] Likewise, in general, therefore, such methods for identifyingcompounds that bind to a polypeptide of the invention can comprisecontacting a compound with a polypeptide of the invention for a timesufficient to form a polypeptide/compound complex, and detecting thecomplex, so that if a polypeptide/compound complex is detected, acompound that binds to a polynucleotide of the invention is identified.

[0370] Methods for identifying compounds that bind to a polypeptide ofthe invention can also comprise contacting a compound with a polypeptideof the invention in a cell for a time sufficient to form apolypeptide/compound complex wherein the complex drives expression of areceptor gene sequence in the cell, and detecting the complex bydetecting reporter gene sequence expression, so that if apolypeptide/compound complex is detected, a compound that binds apolypeptide of the invention is identified.

[0371] Compounds identified via such methods can include compounds whichmodulate the activity of a polypeptide of the invention (that is,increase or decrease its activity, relative to activity observed in theabsence of the compound). Alternatively, compounds identified via suchmethods can include compounds which modulate the expression of apolynucleotide of the invention (that is, increase or decreaseexpression relative to expression levels observed in the absence of thecompound). Compounds, such as compounds identified via the methods ofthe invention, can be tested using standard assays well known to thoseof skill in the art for their ability to modulate activity/expression.

[0372] The agents screened in the above assay can be, but are notlimited to, peptides, carbohydrates, vitamin derivatives, or otherpharmaceutical agents. The agents can be selected and screened at randomor rationally selected or designed using protein modeling techniques.

[0373] For random screening, agents such as peptides, carbohydrates,pharmaceutical agents and the like are selected at random and areassayed for their ability to bind to the protein encoded by the ORF ofthe present invention. Alternatively, agents may be rationally selectedor designed. As used herein, an agent is said to be “rationally selectedor designed” when the agent is chosen based on the configuration of theparticular protein. For example, one skilled in the art can readilyadapt currently available procedures to generate peptides,pharmaceutical agents and the like, capable of binding to a specificpeptide sequence, in order to generate rationally designed antipeptidepeptides, for example see Hurby et al., Application of SyntheticPeptides: Antisense Peptides.” In Synthetic Peptides. A User's Guide. W.H. Freeman, NY (1992), pp. 289-307, and Kaspezak et al., Biochemistry28:9230-8 (1989), or pharmaceutical agents, or the like.

[0374] In addition to the foregoing, one class of agents of the presentinvention, as broadly described, can be used to control gene expressionthrough binding to one of the ORFs or EMFs of the present invention. Asdescribed above, such agents can be randomly screened or rationallydesigned/selected. Targeting the ORF or EMF allows a skilled artisan todesign sequence specific or element specific agents, modulating theexpression of either a single ORF or multiple ORFs which rely on thesame EMF for expression control. One class of DNA binding agents areagents which contain base residues which hybridize or form a triplehelix formation by binding to DNA or RNA. Such agents can be based onthe classic phosphodiester ribonucleic acid backbone, or can be avariety of sulfhydryl or polymeric derivatives which have baseattachment capacity.

[0375] Agents suitable for use in these methods usually contain 20 to 40bases and are designed to be complementary to a region of the geneinvolved in transcription (triple helix—see Lee et al., Nucl. Acids Res.6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan et al.,Science 251:1360 (1991 )) or to the mRNA itself (antisense—Okano. J.Neurochem. 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitorsof Gene Expression. CRC Press. Boca Raton. Fla. (1988)). Triplehelix-formation optimally results in a shut-off of RNA transcriptionfrom DNA, while antisense RNA hybridization blocks translation of anmRNA molecule into polypeptide. Both techniques have been demonstratedto be effective in model systems. Information contained in the sequencesof the present invention is necessary for the design of an antisense ortriple helix oligonucleotide and other DNA binding agents.

[0376] Agents which bind to a protein encoded by one of the ORFs of thepresent invention can be used as a diagnostic agent. Agents which bindto a protein encoded by one of the ORFs of the present invention can beformulated using known techniques to generate a pharmaceuticalcomposition.

4.16 Use of Nucleic Acids as Probes

[0377] Another aspect of the subject invention is to provide forpolypeptide-specific nucleic acid hybridization probes capable ofhybridizing with naturally occurring nucleotide sequences. Thehybridization probes of the subject invention may be derived from any ofthe nucleotide sequences SEQ ID NO: 1-22, 24, 26-27, 29, or 33. Becausethe corresponding gene is only expressed in a limited number of tissues,a hybridization probe derived from of any of the nucleotide sequencesSEQ ID NO: 1-22, 24, 26-27, 29, or 33 can be used as an indicator of thepresence of RNA of cell type of such a tissue in a sample.

[0378] Any suitable hybridization technique can be employed, such as,for example, in situ hybridization. PCR as described in U.S. Pat. Nos.4,683,195 and 4,965,188 provides additional uses for oligonucleotidesbased upon the nucleotide sequences. Such probes used in PCR may be ofrecombinant origin, may be chemically synthesized, or a mixture of both.The probe will comprise a discrete nucleotide sequence for the detectionof identical sequences or a degenerate pool of possible sequences foridentification of closely related genomic sequences.

[0379] Other means for producing specific hybridization probes fornucleic acids include the cloning of nucleic acid sequences into vectorsfor the production of mRNA probes. Such vectors are known in the art andare commercially available and may be used to synthesize RNA probes invitro by means of the addition of the appropriate RNA polymerase as T7or SP6 RNA polymerase and the appropriate radioactively labelednucleotides. The nucleotide sequences may be used to constructhybridization probes for mapping their respective genomic sequences. Thenucleotide sequence provided herein may be mapped to a chromosome orspecific regions of a chromosome using well known genetic and/orchromosomal mapping techniques. These techniques include in situhybridization, linkage analysis against known chromosomal markers,hybridization screening with libraries or flow-sorted chromosomalpreparations specific to known chromosomes and the like. The techniqueof fluorescent in situ hybridization of chromosome spreads has beendescribed, among other places, in Verma et al (1988) Human Chromosomes:A Manual of Basic Techniques, Pergamon Press, New York N.Y.

[0380] Fluorescent in situ hybridization of chromosomal preparations andother physical chromosome mapping techniques may be correlated withadditional genetic map data. Examples of genetic map data can be foundin the 1994 Genome Issue of Science (265:1981 t). Correlation betweenthe location of a nucleic acid on a physical chromosomal map and aspecific disease (or predisposition to a specific disease) may helpdelimit the region of DNA associated with that genetic disease. Thenucleotide sequences of the subject invention may be used to detectdifferences in gene sequences between normal, carrier or affectedindividuals.

4.17 Preparation of Support Bound Oligonucleotides

[0381] Oligonucleotides, i.e., small nucleic acid segments, may bereadily prepared by, for example, directly synthesizing theoligonucleotide by chemical means, as is commonly practiced using anautomated oligonucleotide synthesizer.

[0382] Support bound oligonucleotides may be prepared by any of themethods known to those of skill in the art using any suitable supportsuch as glass, polystyrene or Teflon. One strategy is to precisely spotoligonucleotides synthesized by standard synthesizers. Immobilizationcan be achieved using passive adsorption (Inouye & Hondo, 1990 J. ClinMicrobiol 28(6) 1462-72); using UV light (Nagata et al., 1985; Dahlen etal, 1987; Morrissey & Collins, Mol. Cell Probes 1989 3(2) 189-207) or bycovalent binding of base modified DNA (Keller et al. 1988: 1989); allreferences being specifically incorporated herein.

[0383] Another strategy that may be employed is the use of the strongbiotin-streptavidin interaction as a linker. For example, Broude et al,(1994) Proc. Natl. Acad. Sci USA 91(8) 3072-6 describe the use ofbiotinylated probes, although these are duplex probes, that areimmobilized on streptavidin-coated magnetic beads.Streptavidin-coatedbeads may be purchased from Dynal. Oslo. Of course,this same linking chemistry is applicable to coating any surface withstreptavidin. Biotinylated probes may be purchased from various sources,such as, e.g., Operon Technologies (Alameda, Calif.).

[0384] Nunc Laboratories (Naperville. Ill.) is also selling suitablematerial that could be used. Nunc Laboratories have developed a methodby which DNA can be covalently bound to the microwell surface termedCovalink NH. CovaLink NH is a polystyrene surface grafted with secondaryamino groups (>NH) that serve as bridge-heads for further covalentcoupling. CovaLink Modules may be purchased from Nunc Laboratories. DNAmolecules may be bound to CovaLink exclusively at the 5′-end by aphosphoramidate bond, allowing immobilization of more than 1 pmol of DNA(Rasmussen et al., (1991) Anal Biochem 198(1) 138-42.

[0385] The use of CovaLink NH strips for covalent binding of DNAmolecules at the 5′-end has been described (Rasmussen et al., 1991 ). Inthis technology, a phosphoramidate bond is employed (Chu et al., 1983Nucleic Acids 11 (18) 6513-29). This is beneficial as immobilizationusing only a single covalent bond is preferred. The phosphoramidate bondjoins the DNA to the CovaLink NH secondary amino groups that arepositioned at the end of spacer arms covalently grafted onto thepolystyrene surface through a 2 nm long spacer arm. To link anoligonucleotide to CovaLink NH via an phosphoramidate bond, theoligonucleotide terminus must have a 5′-end phosphate group. It is,perhaps, even possible for biotin to be covalently bound to CovaLink andthen streptavidin used to bind the probes.

[0386] More specifically, the linkage method includes dissolving DNA inwater (7.5 ng/ul) and denaturing for 10 min, at 95° C. and cooling onice for 10 min. Ice-cold 0.1 M 1-methylimidazole pH 7.0 (1-MeIm₇), isthen added to a final concentration of 10 mM 1-MeIm₇. A ss DNA solutionis then dispensed into CovaLink NH strips (75 ul/well) standing on ice.

[0387] Carbodiimide 0.2 M1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide(EDC), dissolved in 10 mM1-MeIm₇, is made fresh and 25 ul added per well. The strips areincubated for 5 hours at 50° C. After incubation the strips are washedusing, e.g,. Nunc-Immuno Wash; first the wells are washed 3 times, thenthey are soaked with washing solution for 5 min., and finally they arewashed 3 times (where in the washing solution is 0.4 N NaOH, 0.25% SDSheated to 50° C.).

[0388] It is contemplated that a further suitable method for use withthe present invention is that described in PCT Patent Application WO90/03382 (Southern & Maskos), incorporated herein by reference. Thismethod of preparing an oligonucleotide bound to a support involvesattaching a nucleoside 3′-reagent through the phosphate group by acovalent phosphodiester link to aliphatic hydroxyl groups carried by thesupport. The oligonucleotides then synthesized on the supportednucleoside and protecting groups removed from the syntheticoligonucleotide chain under standard conditions that do not cleave theoligonucleotide from the support. Suitable reagents include nucleosidephosphoramidite and nucleoside hydrogen phosphorate.

[0389] An on-chip strategy for the preparation of DNA probe for thepreparation of DNA probe arrays may be employed. For example,addressable laser-activated photodeprotection may be employed in thechemical synthesis of oligonucleotides directly on a glass surface, asdescribed by Fodor et al, (1991) Science 251(4995) 767-73, incorporatedherein by reference. Probes may also be immobilized on nylon supports asdescribed by Van Ness et al (1991 ) Nucleic Acids Res. 19(12) 3345-50;or linked to Teflon using the method of Duncan & Cavalier (1988) AnalBiochem 169(1 ) 104-8; all references being specifically incorporatedherein.

[0390] To link an oligonucleotide to a nylon support, as described byVan Ness et al, (1991). requires activation of the nylon surface viaalkylation and selective activation of the 5′-amine of oligonucleotideswith cyanuric chloride.

[0391] One particular way to prepare support bound oligonucleotides isto utilize the light-generated synthesis described by Pease et al.,(1994) Proc. Natl. Acad. Sci USA 91(11) 5022-6. These authors usedcurrent photolithographic techniques to generate arrays of immobilizedoligonucleotide probes (DNA chips). These methods, in which light isused to direct the synthesis of oligonucleotide probes in high-density,miniaturized arrays, utilize photolabile 5′-protectedN-acyl-deoxynucleosidephosphoramidites, surface linker chemistry andversatile combinatorial synthesis strategies. A matrix of 256 spatiallydefined oligonucleotide probes may be generated in this manner.

4.18 Preparation of Nucleic Acid Fragments

[0392] The nucleic acids may be obtained from any appropriate source,such as cDNAs, genomic DNA, chromosomal DNA, microdissected chromosomebands, cosmid or YAC inserts, and RNA, including mRNA without anyamplification steps. For example. Sambrook et al, (1989) describes threeprotocols for the isolation of high molecular weight DNA from mammaliancells (p. 9.14-9.23).

[0393] DNA fragments may be prepared as clones in M13, plasmid or lambdavectors and/or prepared directly from genomic DNA or cDNA by PCR orother amplification methods. Samples may be prepared or dispensed inmultiwell plates. About 100-1000 ng of DNA samples may be prepared in2-500 ml of final volume.

[0394] The nucleic acids would then be fragmented by any of the methodsknown to those of skill in the art including, for example, usingrestriction enzymes as described at 9.24-9.28 of Sambrook et al (1989),shearing by ultrasound and NaOH treatment.

[0395] Low pressure shearing is also appropriate, as described bySchriefer et al, (1990) Nucleic Acids Res. 18(24) 7455-6. In thismethod, DNA samples are passed through a small French pressure cell at avariety of low to intermediate pressures. A lever device allowscontrolled application of lows to intermediate pressures to the cell.The results of these studies indicate that low-pressure shearing is auseful alternative to sonic and enzymatic DNA fragmentation methods.

[0396] One particularly suitable way for fragmenting DNA is contemplatedto be that using the two base recognition endonuclease. CviJI, describedby Fitzgerald et al, (1992) Nucleic Acids Res. 20(14)3753-62. Theseauthors described an approach for the rapid fragmentation andfractionation of DNA into particular sizes that they contemplated to besuitable for shotgun cloning and sequencing.

[0397] The restriction endonuclease CviJI normally cleaves therecognition sequence PuGCPy between the G and C to leave blunt ends.Atypical reaction conditions, which alter the specificity of this enzyme(CviJl**), yield a quasi-random distribution of DNA fragments form thesmall molecule pUC19 (2688 base pairs). Fitzgerald et al, (1992)quantitatively evaluated the randomness of this fragmentation strategy,using a CviJI** digest of pUC19 that was size fractionated by a rapidgel filtration method and directly ligated, without end repair, to a lacZ minus M13 cloning vector. Sequence analysis of 76 clones showed thatCviJI** restricts pyGCPy and PuGCPu, in addition to PuGCPy sites, andthat new sequence data is accumulated at a rate consistent with randomfragmentation.

[0398] As reported in the literature, advantages of this approachcompared to sonication and agarose gel fractionation include: smalleramounts of DNA are required (0.2-0.5 ug instead of 2-5 ug); and fewersteps are involved (no preligation, end repair, chemical extraction, oragarose gel electrophoresis and elution are needed).

[0399] Irrespective of the manner in which the nucleic acid fragmentsare obtained or prepared, it is important to denature the DNA to givesingle stranded pieces available for hybridization. This is achieved byincubating the DNA solution for 2-5 minutes at 80-90° C. The solution isthen cooled quickly to 2° C. to prevent renaturation of the DNAfragments before they are contacted with the chip. Phosphate groups mustalso be removed from genomic DNA by methods known in the art.

4.19 Preparation of DNA Arrays

[0400] Arrays may be prepared by spotting DNA samples on a support suchas a nylon membrane. Spotting may be performed by using arrays of metalpins (the positions of which correspond to an array of wells in amicrotiter plate) to repeated by transfer of about 20 nl of a DNAsolution to a nylon membrane. By offset printing, a density of dotshigher than the density of the wells is achieved. One to 25 dots may beaccommodated in 1 mm², depending on the type of label used. By avoidingspotting in some preselected number of rows and columns, separatesubsets (subarrays) may be formed. Samples in one subarray may be thesame genomic segment of DNA (or the same gene) from differentindividuals, or may be different, overlapped genomic clones. Each of thesubarrays may represent replica spotting of the same samples. In oneexample, a selected gene segment may be amplified from 64 patients. Foreach patient, the amplified gene segment may be in one 96-well plate(all 96 wells containing the same sample). A plate for each of the 64patients is prepared. By using a 96-pin device, all samples may bespotted on one 8×12 cm membrane. Subarrays may contain 64 samples, onefrom each patient. Where the 96 subarrays are identical, the dot spanmay be 1 mm² and there may be a 1 mm space between subarrays.

[0401] Another approach is to use membranes or plates (available fromNUNC, Naperville, Ill.) which may be partitioned by physical spacerse.g, a plastic grid molded over the membrane, the grid being similar tothe sort of membrane applied to the bottom of multiwell plates, orhydrophobic strips. A fixed physical spacer is not preferred for imagingby exposure to flat phosphor-storage screens or x-ray films.

[0402] The present invention is illustrated in the following examples.Upon consideration of the present disclosure, one of skill in the artwill appreciate that many other embodiments and variations may be madein the scope of the present invention. Accordingly, it is intended thatthe broader aspects of the present invention not be limited to thedisclosure of the following examples. The present invention is not to belimited in scope by the exemplified embodiments which are intended asillustrations of single aspects of the invention, and compositions andmethods which are functionally equivalent are within the scope of theinvention. Indeed, numerous modifications and variations in the practiceof the invention are expected to occur to those skilled in the art uponconsideration of the present preferred embodiments. Consequently, theonly limitations which should be placed upon the scope of the inventionare those which appear in the appended claims.

[0403] All references cited within the body of the instant specificationare hereby incorporated by reference in their entirety.

5. EXAMPLES Example 1

[0404] Isolation of SEQ ID NO: 1-21 from a cDNA Libraries of Human Cells

[0405] A plurality of novel nucleic acids were obtained from a cDNAlibrary prepared from human fetal liver spleen, ovary, adult brain, lungtumor, spinal cord, cervix, ovary, endothelial cells, umbilical cord,lymphocyte, lung fibroblast, fetal brain, and testis, using standardPCR. sequencing by hybridization sequence signature analysis, and Sangersequencing techniques. The inserts of the library were amplified withPCR using primers specific for vector sequences flanking the inserts.These samples were spotted onto nylon membranes and interrogated witholigonucleotide probes to give sequence signatures. The clones wereclustered into groups of similar or identical sequences and singlerepresentative clones were selected from each group for gel sequencing.The 5′ sequence of the amplified inserts was then deduced using thereverse M13 sequencing primer in a typical Sanger sequencing protocol.PCR products were purified and subjected to fluorescent dye terminatorcycle sequencing. Single-pass gel sequencing was done using a 377Applied Biosystems (ABI) sequencer. These inserts was identified as anovel sequence not previously obtained from this library and notpreviously reported in public databases. These sequences are designatedas SEQ ID NO: 1-21 in the attached sequence listing.

Example 2

[0406] Assemblage of SEQ ID NO: 22 and 24

[0407] The novel nucleic acids (SEQ ID NO: 22 and 24) of the inventionwere assembled from sequences that were obtained from a cDNA library bymethods described in Example 1 above. The final sequences were assembledusing the EST sequences as seed. Then a recursive algorithm was used toextend the seed into an extended assemblage by pulling additionalsequences from Hyseq's database containing EST sequences that belong tothis assemblage. The algorithm terminated when a complete contig wasassembled. Inclusion of component sequences into the assemblage wasbased on a BLASTN hit to the extending assemblage with BLAST scoregreater than 300 and percent identity greater than 95%.

[0408] The nearest neighbor result for the assembled sequence (SEQ IDNO. 22 or 24) was obtained by a FASTA version 3 search against Genpeptrelease 114, using Fastxy algorithm. Fastxy is an improved version ofFASTA alignment which allows in-codon frame shifts. The nearest neighborresult showed the closest homologue for each assemblage from Genpept(and contains the translated amino acid sequences for which theassemblage encodes). The nearest neighbor result is set forth below:Accession Smith-Waterman % No. Description Score Identity Z35597 Unknownweak similarity with 760 36.188 sea squirt nidogen precursor protein(blastp score 71): cDNA EST EMBL:

[0409] Polypeptides were predicted to be encoded by SEQ ID NO: 22 (or24) as set forth below. The polypeptides were predicted using a softwareprogram called FASTY (available from http://fasta.bioch.virginia.edu)which selects a polypeptide based on a comparison of translated novelpolynucleotide to known polypeptides (W. R. Pearson. Methods inEnzymology, 183: 63-98 (1990), herein incorporated by reference).Predicted Predicted end Amino acid composition of the polvpeptideencoded. beginning nucleotide wherein. (A=Alanine. C=Cvsteine.D=Aspartic Acid. nucleotide location E=Glutamic Acid. F=Phenylalanine.G=Glycine. location corresponding H=Histidine. I=Isoleucine. K=Lvsine.L=Leucine. corresponding to last M=Methionine. N=Asparagine. P=Proline.to first amino acid Q=Glutamine. R=Arginine. S=Serine. T=Threonine.amino acid residue of V=Valine. W=Tryptophan. Y=Tvrosine. X=Unknown.residue of amino acid *Stop Codon. /=possible nucleotide deletion. aminoacid segment \=possible nucleotide insertions segment 2669 1388PRVRPRVRTDHNYYISRIYGPSDSASRDLWVNID QMEKDKVKIHGILSNTHRQAARVNLSFDFPFYGHFLREITVATGGFIYTGEVVHRMLTATQYIAPLM ANFDPSVSRNSTVRYFDNGTALVVQWDHVHLQDNYNLGSFTFQATLLMDGRIIFGYKEIPVLVTQIS STNHPVKVGLSDAFVVVHRIQQIPNVRRRTIYEYHRVELQMSKITNISAVEMTPLPTCLQFNRCGPCV SSQIGFNCSWCSKLQRCSSGFDRHRQDWVDSGCPEESKEKMCENTEPVET\FLEPPQP*ERQPPSSGS* LPPE/DAVTSQFPTSLPTEDDTKIALHLKDNGASTDDSAAEKKGGTLHAGLIVGILILVLIVATAILVTV YMYHHPTSAASIFFIERRPSRWPAMKFRRGSGHPAYAEVEPVGEKEGFIVSEQC (SEQ ID NO: 35)

Example 3

[0410] Assemblage of SEQ ID NO: 27

[0411] The novel nucleic acid (SEQ ID NO: 27) of the invention wasinitially assembled from sequences that were obtained from a cDNAlibrary by methods described in Example 1 above. The final sequence wasassembled using the EST sequences as seed. Then a recursive algorithmwas used to extend the seed into an extended assemblage, by pullingadditional sequences from Hyseq's database containing EST sequences thatbelong to this assemblage. The algorithm terminated when a completecontig was assembled. Inclusion of component sequences into theassemblage was based on a BLASTN hit to the extending assemblage withBLAST score greater than 300 and percent identity greater than 95%.

[0412] Using this initial sequence, suitable primers were designed foramplification of ESTs that comprise the initial sequence. The productswere cloned. The DNA was isolated, cut with appropriate restrictionenzymes, ligated, and recloned to generate the full-length contig. Thefull-length product was then cloned and sequenced using 377 AppliedBiosystems (ABI) sequencer. This nucleotide sequence is identical to SEQID NO: 27.

[0413] Alternatively, the full-length stem cell factor-like DNA was PCRamplified using appropriate primers from Marathon-ready spleen cDNAlibrary (Clontech). The primary PCR product was further amplified usingnested PCR primers. The product of the second PCR was sequenced using377 Applied Biosystems (ABI) sequencer. This product is identical to SEQID NO: 27.

Example 4

[0414] Assemblage of SEQ ID NO: 23, 25, and 28

[0415] Using PHRAP (Univ. of Washington), full-length gene cDNAsequences and the corresponding protein sequences were generated fromthe assemblage. Any frame shifts and incorrect stop codons werecorrected by hand editing. During editing, the sequence was checkedusing FASTY and/or BLAST against Genbank (i.e. Genepept release 115).Other computer programs, which may have been used in the editingprocess, were phredPhrap and Consed (University of Washington) anded-ready, ed-ext and cg-zip-2 (Hyseq. Inc.).

[0416] A polypeptide (SEQ ID NO: 28) was predicted to be encoded by SEQID NO: 27 as set forth below. The polypeptide was predicted using asoftware program called BLASTX which selects a polypeptide based on acomparison of translated novel polynucleotide to known polynucleotides.The initial methionine starts at position 123 of SEQ ID NO: 3 and theputative stop codon, TAA, begins at position 1710 of the nucleotidesequence.

[0417] The stem cell growth factor-like polypeptide of SEQ ID NO: 28 isan approximately 529-amino acid protein with a predicted molecular massof approximately 59.2-kDa, unglycosylated. Protein database searcheswith the BLASTP algorithm (Altschul S. F. et al., J. Mol. Evol.36:290-300 (1993) and Altschul S. F. et al., J. Mol. Biol. 21:403-10(1990), herein incorporated by reference) indicate that SEQ ID NO: 28 ishomologous to tumor endothelial marker 7 precursor protein.

[0418]FIG. 2 shows the BLASTX amino acid sequence alignment between theprotein encoded by stem cell growth factor-like polypeptide SEQ ID NO:28 and tumor endothelial marker 7 precursor protein SEQ ID NO: 36 (St.Croix et al, Science, 289, 1197-1201), indicating that the two sequencesshare 72% similarity over 441 amino acid residues and 57% identity overthe same 441 amino acid residues.

[0419] A predicted approximately thirty-residue signal peptide isencoded from approximately residue 1 through residue 30 of SEQ ID NO: 28(SEQ ID NO: 30). The extracellular portion is useful on its own. Thiscan be confirmed by expression in mammalian cells and sequencing of thecleaved product. The signal peptide region was predicted using NeuralNetwork SignalP V1.1 program (Nielsen e; al, (1997) Int. J. Neur. Syst.8, 581) (from Center for Biological Sequence Analysis. The TechnicalUniversity of Denmark), and hydrophobicity analysis using theKyte/Doolittle algorithm (Kyte and Doolittle (1982) J. Mol. Biol. 157,105). One of skill in the art will recognize that the cleavage site maybe different than that predicted by the computer program. SEQ ID NO: 31is the peptide resulting when the signal peptide is removed from SEQ IDNO: 28.

[0420] A predicted approximately twenty eight-residue transmembraneregion is encoded from approximately residue 452 through residue 479 ofSEQ ID NO: 28 (SEQ ID NO: 32). It may be confirmed by expression inmammalian cells. The transmembrane region was predicted using NeuralNetwork SignalP V1.1 program (Nielsen et al, (1997) Int. J. Neur. Syst.8, 581) (from Center for Biological Sequence Analysis. The TechnicalUniversity of Denmark), and hydrophobicity analysis using theKyte/Doolittle algorithm (Kyte and Doolittle (1 982) J. Mol. Biol. 157,105). One of skill in the art will recognize that the transmembraneregion may be different than that predicted by the computer program.

[0421] A polypeptide (SEQ ID NO: 25) was predicted to be encoded by SEQID NO: 24 as set forth below. The polypeptide was predicted using asoftware program called BLASTX which selects a polypeptide based on acomparison of translated novel polynucleotide to known polynucleotides.The initial methionine starts at position 107 of SEQ ID NO:24 and theputative stop codon, TAA, begins at position 1280 of the nucleotidesequence.

[0422] The stem cell growth factor-like polypeptide of SEQ ID NO: 25(identical to SEQ ID NO: 23) is an approximately 392-amino acid proteinwith a predicted molecular mass of approximately 50-kDa unglycosylated.Protein database searches with the BLASTP algorithm (Altschul S. F. etal., J. Mol. Evol. 36:290-300 (1993) and Altschul S. F. et al., J. Mol.Biol. 21:403-10 (1990), herein incorporated by reference) indicate thatSEQ ID NO: 25 is homologous to tumor endothelial marker 7 precursorprotein.

[0423] A predicted approximately twenty eight-residue transmembraneregion is encoded from approximately residue 315 through residue 342 ofSEQ ID NO: 25 (SEQ ID NO: 32). It may be confirmed by expression inmammalian cells. The transmembrane region was predicted using NeuralNetwork SignalP V1.1 program (Nielsen et al, (1997) Int. J. Neur. Svst.8, 58 1) (from Center for Biological Sequence Analysis. The TechnicalUniversity of Denmark), and hydrophobicity analysis using theKyte/Doolittle algorithm (Kyte and Doolittle (1982) J. Mol. Biol. 157,105). One of skill in the art will recognize that the transmembraneregion may be different than that predicted by the computer program.

Example 5

[0424] A. Cloning and Expression of Soluble Stem Cell Factor-LikePolynucleotide (SEQ ID NO: 33) and Polypeptide (SEQ ID NO: 34)

[0425] In order to express soluble stem cell factor-like polypeptide,the full-length stem cell factor-like DNA was PCR amplified fromMarathon-ready spleen cDNA library (Clontech). The primary PCR productwas further amplified using nested PCR primers, that would generatesoluble stem cell factor-like polypeptide when expressed in suitablecell lines. The product of the secondary PCR (SEQ ID NO: 33) was clonedin pCDNA3.1/Myc-His (+) A between EcoRI and XhoI sites. The plasmidencoding soluble stem cell factor-like polypeptide and control vectorswere transfected into CHO cells using FuGENE-6 transfection reagent(Roche). Culture medium, cell lysate and the insoluble cell debrisfractions were analyzed by SDS-PAGE followed by western blotting withanti myc antibodies. As expected, more than 95% of the soluble stem cellfactor-like polypeptide (SEQ ID NO: 34) was found to be secreted andpresent in the culture medium.

[0426] Using similar approach, stable lines of 293 cells expressing SEQID NO: 34 are also generated. These were further cloned to select high,moderate and low expressors.

[0427] B. Expression and Purification of SEQ ID NO: 34 from Insect andBacterial Cells

[0428] Stem cell factor-like protein was expressed in insect cells asfollows:

[0429] The C-terminal transmembrane domain truncated version of stemcell factor-like gene (SEQ ID NO: 33) was cloned by PCR into apIB/V5-His TOPO TA cloning vector (Invitrogen Corporation). The stemcell factor-like DNA in the vector was generated either with a Myc/Histag or without any tags. Insect cells (High Five TM. Invitrogen) weretransfected with the stem cell growth factor-like plasmid DNA containingthe tag by using the InsectSelectTM System (Invitrogen). The expressionof the stem cell growth factor-like protein was determined by transientexpression. The medium containing expressed stem cell growth factor-likeprotein was separated on SDS-PAGE and stem cell growth factor-likeprotein was identified by Western blot analysis. For large-scaleproduction of stem cell growth factor-like protein, resistant cells wereexpanded into flasks containing Ultimate Insect TM Serum-Free medium(Invitrogen). The cells were shaken at ˜100 mph at 27° C. for 4 days.The conditioned media containing the protein for purification werecollected by centrifugation.

[0430] Stem cell factor-like protein was expressed in bacterial cells asfollows:

[0431] The mature stem cell growth factor-like gene without thetransmembrane domain (SEQ ID NO: 33) was cloned into an expressionvector (PCR T7/NT-TOPO) from Invitrogen. The resulting plasmid wasexpressed in E.coli BL-21 (DE 3) pLys strain. Cells were grown in LBbroth containing ampicillin (100 μg/mL) at 37° C. Expression of stemcell growth factor-like protein was then induced with IPTG (1 mM finalconcentration), and cells were grown for an additional 4 hours andharvested. Analysis of stem cell growth factor-like production bySDS-PAGE and Western blotting was done as detailed above.

[0432] Purification of stem cell growth factor-like protein from insectcell cultures was carried out as follows. Insect Ultimate mediumcontaining the His-tagged stem cell growth factor-like was to pH 7.5 byadding appropriate quantity of 1M NaOH. The solution was thensupplemented with 1 mM PMSF (final concentration) to prevent theproteolytic cleavage during the purification process. The medium waspassed through a 0.2 micron filter (Nalgene Surfactant Free CelluloseAcetate 1000 mL sterile filter unit) to remove particulate material. Theresulting solution was concentrated 10-fold and simultaneouslyequilibrated with 20 mM sodium phosphate, pH 7.5 using a diafiltrationcartridge with a membrane cut off size of 10 kDa. The 10-foldconcentrated and diafiltered media was loaded onto a Ni-NTA columnequilibrated with 20 mM sodium phosphate, pH 7.5. Unretained componentsWere removed by, washing the column with 20 mM sodium phosphate pH 7.5containing 300 mM NaCl and 20 mM Imidazole. The His-tagged stem cellgrowth factor-like protein was eluted with the same buffer and a lineargradient of imidazole (20-300 mM). The eluted protein was identified asdescribed above. The pooled fractions containing stem cell growthfactor-like were equilibrated with PBS buffer using Amicon stircell witha membrane cut off size of 10 kDa. This process also resulted in theremoval of imidazole. The protein was then concentrated to approximately10 mg/mL in PBS buffer for functional studies.

[0433] Purification of stem cell growth factor-like protein frombacterial cultures was carried out as follows. E. Coli cells expressingstem cell growth factor-like as inclusion bodies were extracted with 10volumes (wt/vol) of extraction buffer (50 mM NaPO4, pH 7.0) and furtherwith buffer containing 6M guanidine hydrochloride in the extractionbuffer. The solubilized stem cell growth factor-like protein wasfractionated on a Ni-NTA column as described above. The unfolded versionof stem cell growth factor-like protein obtained from this affinitypurification was allowed to attain a native conformation by incubationwith a refolding buffer consisting of DTT and glutathione. Refoldedsample was equilibrated with 20mM Tris. 0.1% Tween and concentrated to100 mL (10×conc.) prior to fast-flow liquid chromatography onion-exchangers Q-sepharose and SP-sepharose. Additional protocols werealso developed for appropriate refolding conditions using 8M ureainstead of 6M guanidine hydrochloride.

Example 6

[0434] Expression of SEQ ID NO: 33 in Primary Human Cells

[0435] The product of the secondary nested PCR from Marathon spleenlibrary or any other polynucleotide encoding stem cell growthfactor-like polypeptide are cloned into MSCV retroviral vector(Clontech) into suitable cloning sites using appropriate forward andreverse PCR primers. This retroviral vector is then transfected usingFUGENE-6 transfection reagent into packaging cell lines to producesuitably large quantities of retrovirus that will have the stem cellgrowth factor-like DNA cloned in it. Retrovirus containing supernatantsare prepared from packaged cell lines and mixed with stromal or stemcells. Upon retrovirus transduction these transduced cells may expressthe stem cell growth factor-like protein which can then be analyzed asfollows:

[0436] A. Liquid Culture Assay: Stem cells from hematopoietic or otherorigins are commercially purchased. 1×10⁴ stem cells will be plated in a96-well plate. 50-200 ng/ml of purified stem cell growth factor-likeprotein or other suitable growth factors at appropriate concentrationswill be added to the stem cells. IL-3 and IL-6 will be added after 5days of incubation. Cultures are microscopically observed and countedevery day. Flow cytometry staining is performed to determine celllineage differentiation.

[0437] B. Stroma-associated Culture Assay: Stromal cells from suitabletissues are obtained from commercial vendors. 1×10⁴ stem cells will beco-cultured with 1×10⁴ stem cell growth factor-like polynucleotidetransduced stromal cells. Cultures are microscopically observed andcounted every day. Flow cytometry staining can be performed to determinecell lineage differentiation.

Example 7

[0438] Expression Study Using SEQ ID NO: 1-22, 24, 26-27, 29, or 33

[0439] The expression of SEQ ID NO: 1-22, 24, 26-27, 29, or 33 invarious tissues is analyzed using a semi-quantitative polymerase chainreaction-based technique. Human cDNA libraries are used as sources ofexpressed genes from tissues of interest (adult bladder, adult brain,adult heart, adult kidney, adult lymph node, adult liver, adult lung,adult ovary, adult placenta, adult rectum, adult spleen, adult testis,bone marrow, thymus, thyroid gland, fetal kidney, fetal liver, fetalliver-spleen, fetal skin, fetal brain, fetal leukocyte and macrophage).Gene-specific primers are used to amplify portions of SEQ ID NO: 1-22,24, 26-27, 29, or 33 sequences from the samples. Amplified products areseparated on an agarose gel, transferred and chemically linked to anylon filter. The filter is then hybridized with a radioactively labeled(³³P-dCTP) double-stranded probe generated from SEQ ID NO: 1-22, 24,26-27, 29, or 33 using a Klenow polymerase, random-prime method. Thefilters are washed (high stringency) and used to expose aphosphorimaging screen for several hours. Bands indicate the presence ofcDNA including SEQ ID NO: 1-22, 24, 26-27, 29, or 33 sequences in aspecific librant, and thus mRNA expression in the corresponding celltype or tissue.

1 36 1 366 DNA Homo sapiens 1 ggcacgagct acatctaaaa gataatggagcttctacaga tgacagtgca gctgagaaga 60 aagggggaac cctccacgct ggcctcatcgttggaatcct catcctggtc ctcattgtag 120 ccacagccat tcttgtgaca gtctatatgtatcaccaccc aacatcagca gccagcatct 180 tctttattga gagacgccca agcagatggcctgcgatgaa gtttagaaga ggctctggac 240 atcctgccta tgctgaagtt gaaccagttggagagaaaga aggctttatt gtatcagagc 300 agtgctaaaa tttctaggac agaacaacaccagtactggt ttacaggtgt taagactaaa 360 attttg 366 2 334 DNA Homo sapiens 2ggcacgagct acatctaaca gataatggag cttctacaga tgacagggca gctgagaaga 60aagggggaac cctccacgct ggcctcatcg ttggaatcct catcctggtc ctcattgtag 120ccacagccat tcttgtgaca gtctatatgt atcaccaccc aacatcagca gccagcatct 180tctttattga gagacgccca agcagatggc ctgcgatgaa gtttagaaga ggctctggac 240atcctgccta tgctgaagtt gaaccagttg gagagaaaga aggctttatt gtatcagagc 300agtgctaaaa tttctaggac agaacaacac cagt 334 3 422 DNA Homo sapiens 3cagaaattca actgtcagat attttgataa tggcacagca cttgtggtcc agtgggacca 60tgtacatctc caggataatt ataacctggg aagcttcaca ttccaggcaa ccctgctcat 120ggatggacga atcatctttg gatacaaaga aattcctgtc ttggtcacac agataagttc 180aaccaatcat ccagtgaaag tcggactgtc cgatgcattt gtcgttgtcc acaggatcca 240acaaattccc agtacgtaga agaagggcag tcgcaatgag tgagcctctg tgggggtaaa 300tttaaaggag attggtctat ggcagctgta cctgaattaa aaaaaaaata gctaatcgat 360tagctgatta atgcttaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaag 420 gg422 4 460 DNA Homo sapiens misc_feature (21) n = A, T, G, or C 4ggcgggaaac tcgatgacca ntagnaagtt cganngccat tagacagtgc ggaggaattc 60aaccctttca ctcaaaagag caatgatgaa tgtctcatga tagctaagaa caactagccc 120atgcaagagt gagaacaaac acaaaataag agattttcta cattttcaaa acagatgtgt 180ggcaaaagga tgttgttttt ctggtctaga tccatctgta ccaacaagtt catcacttta 240cagaacgaat ctttttatcc gtacaggagg ttcaaaccat gtctgcctct tcctttgtaa 300tgaatgacct ttctatgagc tgtgacaaaa tttccgaaca attagctaag gatttgggaa 360gagggggtgg caaacggggc tttctgtttt cctgcctcag catgaaaaca tctgatttat 420gctttatgga agccttacct ccaatcccca actgttaaan 460 5 447 DNA Homo sapiens 5cggaacatct cccgtggact tatctgaagt atgacaagat tataatgctt ttggcttaag 60cgcagggttg caaagggatc acaaaaaaaa aatcataata aagctttagt tcatgaggga 120tcgaaaaaaa caacaaaaaa aacaaaactg aaataactct ataaaaaaaa aaaaaaagaa 180aggtaatgac ttacttttga aaggaataac acactgcctg aaaaaagacc acaaagacct 240ggcccaaatt cagaactgtg ttagtgcgga tctcccccca gtctcaacat taggaggctc 300ctcattcttt gggagatatg aaaacataaa tggagctgtt aacaagggaa ccgcccagaa 360aatgtgggtt cacctgcaag accaccccca ccattttgtc tctacgtgcc cttgtggata 420gtgaatcgct tcattccaac tcccact 447 6 484 DNA Homo sapiens misc_feature(17) n = A, T, G, or C 6 gagggcattg aatgatnacc ctaggccagn gcggnggaattcgtttacag gtgttaagac 60 taaaattttg cctatacctt taagacaaac aaactaacactcacacaaac aagctctaag 120 ctgctgtagc ctgaagaaga caagatttct ggacaagctcagcccaggaa acaaagggta 180 aacaaaaaac taaaacttat acaagatacc atttacactgaacatagaat tccctagtgg 240 aatgtcatct atagttcact cggaacatct cccgtggacttatctgaagt atgacaagat 300 tataatgctt ttggcttagg tgcagggttg caaagggatcagaaaaaaaa atcataataa 360 agctttagtt catgagggat cgacaccttt ggttcaaatgttctctgatg tctcaaagat 420 aactgttttc caaagcctga accctttcac tcaaaagagcaatgatgaat gtctcaagat 480 tgct 484 7 498 DNA Homo sapiens misc_feature(7) n = A, T, G, or C 7 gcggggnnat tgaaaccttg gagatcgaga ccctagtcagngtgcnggaa ttccacagat 60 aagttcaacc aatcatccag tgaaagtcgg actgtccgatgcatttgtcg ttgtccacag 120 gatccaacaa attcccaatg ttcgaagaag aacaatttatgaataccacc gagtagagct 180 acaaatgtca aaaattacca acatttcggc tgtggagatgaccccattac ccacatgcct 240 ccagtttaac agatgtggcc cctgtgtatc ttctcagattggcttcaact gcagttggtg 300 tagtaaactt caaagatgtt ccagtggatt tgatcgtcatcggcaggact gggtggacag 360 tggatgccct gaagagtcaa aagagaagat gtgtgagaatacagaaccag tggaaacttc 420 ttctcgaacc accacaacca taggagcgac aaccacccagttcagggtcc taactaccac 480 cagaagagca gtgacttt 498 8 405 DNA Homo sapiens8 ggcgaccgac gcgtccgcgg acgcgtgggg aagaggttgt ggcaaacggt tctttctgtt 60ttcctgcctc agcatgaaaa catctgattt atgctttatg gaagccttac ctccaatccc 120caactgttaa gtcccatgaa accacagttg ctctgggctg atggaaacaa aaggaaacag 180tatgaagagt tccttaatca tttttgaaac aaaaatgtta agggatttta aacatatgat 240tatttttaat tttatgcctt ttcagtacta aacacccatt tcattgctga ttcctggcta 300agaagccatt cacgtcagca tggcgataga aagaatgaaa aaaccctgct gaatcataca 360gtaattttct ttaaagcaca tagtagctac ataaatatat atatt 405 9 407 DNA Homosapiens 9 ggggaaaagg ggggggcaaa cggggctttc tgttttcctg gctcagcatgaaaacatctg 60 atttatgctt tatggaagcc ttacctccaa tccccaactg ttaagtcccatgaaaccaca 120 gttgctctgg gctgatggaa acaaaaggaa acagtatgaa gagttccttaatcatttttg 180 aaacaaaaat gttaagggat tttaaacata tgattatttt taattttatgccttttcagt 240 actaaacacc catttcattg ctgattcctg tctaaaaagc cattcacgtcagcatggcga 300 tagaaagaaa gaaaaaaccc tgctgaatca tacagtaatt ttctttaaagcacatagtag 360 ctacataaat atatatatat aaatatattt ttgtttataa ctaacac 40710 392 DNA Homo sapiens 10 aatacactgg ggccacatct gttaaactgg atgcatgtgggtaatggggt catctccaca 60 gccgaaatgt tggtaatttt tgacatttgt agctctacccggtggtattc ataaattgtt 120 cttcttcgaa cattgggaat ttgttggatc ctgtggacaacgacaaatgc atcggacagt 180 ccgactttca ctggatgatt ggttgaactt atctgtgtgaccaagacagg aatttctttg 240 tatccaaaga tgattcgtcc atccatgagc agggttgcctggaatgtgaa gcttcccagg 300 ttataattat cctggagatg tacatggtcc cactggaccacaagtgctgt gccattatca 360 aaatatctga cagttgaatt tctggataca ct 392 11 417DNA Homo sapiens 11 aatacatggt ggtgatacat atagactgtc acaagaatggctgtggctac aatgaggacc 60 aggatgagga ttccaacgat gaggccagcg tggagggttccccctttctt ctcagctgca 120 ctgtcatctg tagaagctcc attatctttt agatgtagtgctatcttggt atcatcttct 180 gtagggaggc tggtgggaaa ctgagaagtc actgctcttctggtggtagt taggaccctg 240 aactgggtgg ttgtcgctcc tatggttgtg gtggttcgagaagaagtttc cactggttct 300 gtattctcac acatcttctc ttttgactct tcagggcatccactgtccac ccagtcctgc 360 cgatgacgat caaatccact ggaacatctt tgaagtttactacaccaact gcagttg 417 12 415 DNA Homo sapiens 12 cggacgcgtg ggtcgaatgctaacagccac agtacatagc acctttaatg gcaaatttcg 60 atcccagtgt atccagaaattcaactgtca gatattttga taatggcaca gcacttgtgg 120 tccagtggga ccatgtacatctccaggata attataacct gggaagcttc acattccagg 180 caaccctgct catggatggacgaatcatct ttggatacaa agaaattcct gtcttggtca 240 cacagataag ttcaaccaatcatccagtga aagtcggact gtccgatgca tttgtcgttg 300 tccacaggat ccaacaaattcccagtacgt aaaagaaggg cagtcgcaat gagtgagcct 360 ctgtgggggt aaatttaaaggagattggtc tatggcagct gtacctgaat taaaa 415 13 494 DNA Homo sapiens 13ccgtcagtgt ggaggaattc gcaagagtga atctgtcctt cgattttcca tattatgggc 60acttcctacg tgaaatcact gtggcaaccg gcggtttcat atacactgga gaagtcgcac 120atcgaaggct aacaaccaca cagtacatag cacctttaat aggcaaatat cgatcccagt 180gtatccagaa attcatctga cagatatttt gataatggca cagcacttgt ggtccagtgg 240gaccatgtac atcttcagga taattataac ctgggaagct tgacattcca ggcgaccctg 300ctcatggatg gacgaatcat ctttggatac aaagaaattc ctgtcttggt cacacagatc 360agttcaacca atcatccagt gaaagtcgga ctgtccgatg catttgtcgt tgtccacagg 420atccaacaaa ttcccaatgt tcgaagaaga acaatttatg aataccaccg agtagagcta 480caaatgtcga acat 494 14 453 DNA Homo sapiens 14 aagatttcta ggacagaacaacaccagtac tggtttacag gtgttaagac taaaattttg 60 cctatacctt taagacaaacaaacaaacac acacacaaac aagctctaag ctgctgtagc 120 ctgaagaaga caagatttctggacaagctc agcccaggaa acaaagggta aacaaaaaac 180 taaaacttat acaagataccatttacactg aacatagaat tccctagtgg aatgtcatct 240 atagttcact cggaacatctcccgtggact tatctgaagt atgacaagat tataatgctt 300 ttggcttagg tgcagggttgcaaagggatc agaaaaaaaa aatcataata aagctttagt 360 tcatgaggga tcgacacctttggttcaaat gttctctgat gtctcaaaga taactgtttt 420 ccaaagcctg aaccctttcactcaaaagag caa 453 15 430 DNA Homo sapiens misc_feature (430) n = A, T,G, or C 15 ctgcaggaat tcggcacgag cccaacatca gcagccagca tcttctttattgagagacgc 60 ccaagcagat ggcctgcgat gaagtttaga agaggctctg gacatcctgcctatgctgaa 120 gttgaaccag ttggagagaa agaaggcttt attgtatcag agcagtgctaaaatttctag 180 gacagaacaa caccagtact ggtttacagg tgttaagact aaaattttgcctataccttt 240 aagacaaaca aacaaacaca cacacaaaca agctctaagc tgctgtagcctgaagaagac 300 aagatttctg gacaagctca gcccaggaaa caaagggtaa acaaaaaactaaaacttata 360 caagatacca tttacactga acatagaatt ccctagtgga atgtcatctatagttcactc 420 ggaacatctn 430 16 405 DNA Homo sapiens misc_feature (370)n = A, T, G, or C 16 agagaaagaa ggctttattg tatcagagca gtgctaaaatttctaggaca gaacaacacc 60 agtactggtt tacaggtgtt aagactaaaa ttttgcctatacctttaaga caaacaaaca 120 aacacacaca caaacaagct ctaagctgct gtagcctgaagaagacaaga tttctggaca 180 agctcagccc aggaaacaaa gggtaaacaa aaaactaaaacttatacaag ataccattta 240 cactgaacat agaattccct agtggaatgt catctatagttcactcggaa catctcccgt 300 ggacttatct gaagtatgac aagattataa tgcttttggcttatgtgcag ggttgcaaag 360 ggatcagaan aaaaaaaaaa aaaaaaaaaa aaaagggggggcgtt 405 17 412 DNA Homo sapiens misc_feature (412) n = A, T, G, or C17 cccacgcgtc cgcccacgcg tccgtacaga ccacaattac tatatatctc gaatatatgg 60tccatctgat tctgccagcc gggatttatg ggtgaacata gaccaaatgg aaaaagataa 120agtgaagatt catggaatat tgtccaatac tcatcggcaa gctgcaagag tgaatctgtc 180cttcgatttt ccattttatg gccacttcct acgtgaaatc actgtggcaa ccgggggttt 240catatacact ggagaagtcg tacatcgaat gctaacagcc acacagtaca tagcaccttt 300aatggcaaat ttcgatccca gtgtatccag aaattcaact gtcagatatt ttgataatgg 360cacagcactt gtggtccagt gggaccatgt acatctccag gataattata an 412 18 440 DNAHomo sapiens misc_feature (429) n = A, T, G, or C 18 gaattcggcacgagctctaa gctgctgtag cctgaagaag acaagatttc tggacaagct 60 cagcccaggaaacaaagggt aaacaaaaaa ctaaaactta tacaagatac catttacact 120 gaacatagaattccctagtg gaatgtcatc tatagttcac tcggaacatc tcccgtggac 180 ttatctgaagtatgacaaga ttataatgct tttggcttag gtgcagggtt gcaaagggat 240 cagaaaaaaaaaatcataat aaagctttag ttcatgaggg aaaaaaaaaa aaaaaaaacc 300 tcggggggggcccgggcccc catttcccct tatagggggc ggtataacaa tccctgggcc 360 gcggtttaacaccgccggga cgggaaaacc cctggggtac cccacttaaa tccctttgga 420 caaaaaaannannagggcgg 440 19 416 DNA Homo sapiens 19 gttgccgatg gggaagaatcacagcggccg caatacatgg gtctgtattc tcacacatct 60 tttcttttga ctcttcagggcatccactgt ccacggggtc cttgtcgatg acgatcaaat 120 ccactggaac atctttgaagtttactacac caactgcagt tgaagccaat ctgagaagat 180 acacaggggc cacatctgttaaactggagg catgtgggta atggggtcat ctccacagcc 240 gaaatgttgg taatttttgacatttgtagc tctactcggt ggtattcata aattgttctt 300 ctttcaacat tgggaatttgctggatcctg gggacaacga caaatgcatt ggacaggccg 360 actttcactg gatgaatggatgaacttatc tgggggagca agacaggaat ttcttg 416 20 382 DNA Homo sapiens 20aatacatggt tctgattctc acacatcttc tcttttgact cttcagggca tccactgtcc 60acccagtccc gccgatgacg atcaaatcca ctggaacatc tttgaagttt actacaccaa 120ctgcagttga agccaatctg agaagataca caggggccac atctgttaaa ctggaggcat 180gtgggtaatg gggtcatctc cacagccgaa atgttggtaa tttttgacat ttgtagctct 240actcggtggt attcataaat tgttcttctt cgaacattgg gaatttgttg gatcctgtgg 300acaacgacaa atgcatcgga cagtccgact ttcactggat gattgggtga acttatctgt 360gggaccaaga caggaatttc tt 382 21 406 DNA Homo sapiens 21 aatacatgcctggaatgtga agcttcccag gttataatta tcctggagat gtacatggtc 60 ccactggaccacaagtgctg tgccattatc aaaatatctg acagttgaat ttctggatac 120 actgggatcgaaatttgcca ttaaaggtgc tatgtactgt gtggctgtta gcattcgatg 180 tacgacttctccagtgtata tgaaaccccc ggttgccaca gtgatttcac gtaggaagtg 240 gccataaaatggaaaatcga aggacagatt cactcttgca gcttgccgat gagtattgga 300 caatattccatgaatcttca ctttatcttt ttccatttgg tctatgttca cccataaatc 360 ccggctggcagaatcagatg gaccatatat tcgagatata tagtag 406 22 2668 DNA Homo sapiens 22cccacgcgtc cgcccacgcg tccgtacaga ccacaattac tatatatctc gaatatatgg 60tccatctgat tctgccagcc gggatttatg ggtgaacata gaccaaatgg aaaaagataa 120agtgaagatt catggaatat tgtccaatac tcatcggcaa gctgcaagag tgaatctgtc 180cttcgatttt ccattttatg gccacttcct acgtgaaatc actgtggcaa ccgggggttt 240catatacact ggagaagtcg tacatcgaat gctaacagcc acacagtaca tagcaccttt 300aatggcaaat ttcgatccca gtgtatccag aaattcaact gtcagatatt ttgataatgg 360cacagcactt gtggtccagt gggaccatgt acatctccag gataattata acctgggaag 420cttcacattc caggcaaccc tgctcatgga tggacgaatc atctttggat acaaagaaat 480tcctgtcttg gtcacacaga taagttcaac caatcatcca gtgaaagtcg gactgtccga 540tgcatttgtc gttgtccaca ggatccaaca aattcccaat gttcgaagaa gaacaattta 600tgaataccac cgagtagagc tacaaatgtc aaaaattacc aacatttcgg ctgtggagat 660gaccccatta cccacatgcc tccagtttaa cagatgtggc ccctgtgtat cttctcagat 720tggcttcaac tgcagttggt gtagtaaact tcaaagatgt tccagtggat ttgatcgtca 780tcggcaggac tgggtggaca gtggatgccc tgaagagtca aaagagaaga tgtgtgagaa 840tacagaacca gtggaaactt cttctcgaac caccacaacc ataggagcga caaccaccca 900gttcagggtc ctaactacca ccagaagagc agtgacttct cagtttccca ccagcctccc 960tacagaagat gataccaaga tagcactaca tctaaaagat aatggagctt ctacagatga 1020cagtgcagct gagaagaaag ggggaaccct ccacgctggc ctcatcgttg gaatcctcat 1080cctggtcctc attgtagcca cagccattct tgtgacagtc tatatgtatc accacccaac 1140atcagcagcc agcatcttct ttattgagag acgcccaagc agatggcctg cgatgaagtt 1200tagaagaggc tctggacatc ctgcctatgc tgaagttgaa ccagttggag agaaagaagg 1260ctttattgta tcagagcagt gctaaaattt ctaggacaga acaacaccag tactggttta 1320caggtgttaa gactaaaatt ttgcctatac ctttaagaca aacaaacaaa cacacacaca 1380aacaagctct aagctgctgt agcctgaaga agacaagatt tctggacaag ctcagcccag 1440gaaacaaagg gtaaacaaaa aactaaaact tatacaagat accatttaca ctgaacatag 1500aattccctag tggaatgtca tctatagttc actcggaaca tctcccgtgg acttatctga 1560agtatgacaa gattataatg cttttggctt aggtgcaggg ttgcaaaggg atcagaaaaa 1620aaaaatcata ataaagcttt agttcatgag ggatcgacac ctttggttca aatgttctct 1680gatgtctcaa agataactgt tttccaaagc ctgaaccctt tcactcaaaa gagcaatgat 1740gaatgtctca agattgctaa gaaaaacagc ccatgcaaga gtgagaacaa acacaaaata 1800agagattttc tacattttca aaacagatgt gtggcaaaag gatgttgttt ttctggtcta 1860gatccatctg taccaacaag ttcatcactt tacagaacga atctttttat ccgtacagga 1920ggttcaaacc atgtctgcct cttcctttgt aatgaatgac ctttctatga gctgtgacaa 1980aatttccgaa caattagcta aggatttggg aagagggggt ggcaaacggg gctttctgtt 2040ttcctgcctc agcatgaaaa catctgattt atgctttatg gaagccttac ctccaatccc 2100caactgttaa gtcccatgaa accacagttg ctctgggctg atggaaacaa aaggaaacag 2160tatgaagagt tccttaatca tttttgaaac aaaaatgtta agggatttta aacatatgat 2220tatttttaat tttatgcctt ttcagtacta aacacccatt tcattgctga ttcctgtcta 2280agaagccatt cacgtcagca tggcgataga aagaatgaaa aaaccctgct gaatcataca 2340gtaattttct ttaaagcaca tagtagttac ataaatatat atatataaat atatttttgt 2400ttataactaa cacaaggcag gatcttgtga ctctaagagt gcgttttgtc atcaagacaa 2460aacagatgca agatgcatca ctgcattact tccatagagt tgtaaaataa tccttaatat 2520tagaatattt ttctgtcact tagcaaaagt ggttcagttc attgccgcgc ccatcatgtt 2580cttgactatt tgatccactt tttcgtttat gtcaacccct tccctctctg gctaaataaa 2640gtggatgcag aaagctcctt aaatggaa 2668 23 392 PRT Homo sapiens 23 Met GluLys Asp Lys Val Lys Ile His Gly Ile Leu Ser Asn Thr His 1 5 10 15 ArgGln Ala Ala Arg Val Asn Leu Ser Phe Asp Phe Pro Phe Tyr Gly 20 25 30 HisPhe Leu Arg Glu Ile Thr Val Ala Thr Gly Gly Phe Ile Tyr Thr 35 40 45 GlyGlu Val Val His Arg Met Leu Thr Ala Thr Gln Tyr Ile Ala Pro 50 55 60 LeuMet Ala Asn Phe Asp Pro Ser Val Ser Arg Asn Ser Thr Val Arg 65 70 75 80Tyr Phe Asp Asn Gly Thr Ala Leu Val Val Gln Trp Asp His Val His 85 90 95Leu Gln Asp Asn Tyr Asn Leu Gly Ser Phe Thr Phe Gln Ala Thr Leu 100 105110 Leu Met Asp Gly Arg Ile Ile Phe Gly Tyr Lys Glu Ile Pro Val Leu 115120 125 Val Thr Gln Ile Ser Ser Thr Asn His Pro Val Lys Val Gly Leu Ser130 135 140 Asp Ala Phe Val Val Val His Arg Ile Gln Gln Ile Pro Asn ValArg 145 150 155 160 Arg Arg Thr Ile Tyr Glu Tyr His Arg Val Glu Leu GlnMet Ser Lys 165 170 175 Ile Thr Asn Ile Ser Ala Val Glu Met Thr Pro LeuPro Thr Cys Leu 180 185 190 Gln Phe Asn Arg Cys Gly Pro Cys Val Ser SerGln Ile Gly Phe Asn 195 200 205 Cys Ser Trp Cys Ser Lys Leu Gln Arg CysSer Ser Gly Phe Asp Arg 210 215 220 His Arg Gln Asp Trp Val Asp Ser GlyCys Pro Glu Glu Ser Lys Glu 225 230 235 240 Lys Met Cys Glu Asn Thr GluPro Val Glu Thr Ser Ser Arg Thr Thr 245 250 255 Thr Thr Ile Gly Ala ThrThr Thr Gln Phe Arg Val Leu Thr Thr Thr 260 265 270 Arg Arg Ala Val ThrSer Gln Phe Pro Thr Ser Leu Pro Thr Glu Asp 275 280 285 Asp Thr Lys IleAla Leu His Leu Lys Asp Asn Gly Ala Ser Thr Asp 290 295 300 Asp Ser AlaAla Glu Lys Lys Gly Gly Thr Leu His Ala Gly Leu Ile 305 310 315 320 ValGly Ile Leu Ile Leu Val Leu Ile Val Ala Thr Ala Ile Leu Val 325 330 335Thr Val Tyr Met Tyr His His Pro Thr Ser Ala Ala Ser Ile Phe Phe 340 345350 Ile Glu Arg Arg Pro Ser Arg Trp Pro Ala Met Lys Phe Arg Arg Gly 355360 365 Ser Gly His Pro Ala Tyr Ala Glu Val Glu Pro Val Gly Glu Lys Glu370 375 380 Gly Phe Ile Val Ser Glu Gln Cys 385 390 24 2668 DNA Homosapiens CDS (107)..(1282) 24 cccacgcgtc cgcccacgcg tccgtacaga ccacaattactatatatctc gaatatatgg 60 tccatctgat tctgccagcc gggatttatg ggtgaacatagaccaa atg gaa aaa 115 Met Glu Lys 1 gat aaa gtg aag att cat gga ata ttgtcc aat act cat cgg caa gct 163 Asp Lys Val Lys Ile His Gly Ile Leu SerAsn Thr His Arg Gln Ala 5 10 15 gca aga gtg aat ctg tcc ttc gat ttt ccattt tat ggc cac ttc cta 211 Ala Arg Val Asn Leu Ser Phe Asp Phe Pro PheTyr Gly His Phe Leu 20 25 30 35 cgt gaa atc act gtg gca acc ggg ggt ttcata tac act gga gaa gtc 259 Arg Glu Ile Thr Val Ala Thr Gly Gly Phe IleTyr Thr Gly Glu Val 40 45 50 gta cat cga atg cta aca gcc aca cag tac atagca cct tta atg gca 307 Val His Arg Met Leu Thr Ala Thr Gln Tyr Ile AlaPro Leu Met Ala 55 60 65 aat ttc gat ccc agt gta tcc aga aat tca act gtcaga tat ttt gat 355 Asn Phe Asp Pro Ser Val Ser Arg Asn Ser Thr Val ArgTyr Phe Asp 70 75 80 aat ggc aca gca ctt gtg gtc cag tgg gac cat gta catctc cag gat 403 Asn Gly Thr Ala Leu Val Val Gln Trp Asp His Val His LeuGln Asp 85 90 95 aat tat aac ctg gga agc ttc aca ttc cag gca acc ctg ctcatg gat 451 Asn Tyr Asn Leu Gly Ser Phe Thr Phe Gln Ala Thr Leu Leu MetAsp 100 105 110 115 gga cga atc atc ttt gga tac aaa gaa att cct gtc ttggtc aca cag 499 Gly Arg Ile Ile Phe Gly Tyr Lys Glu Ile Pro Val Leu ValThr Gln 120 125 130 ata agt tca acc aat cat cca gtg aaa gtc gga ctg tccgat gca ttt 547 Ile Ser Ser Thr Asn His Pro Val Lys Val Gly Leu Ser AspAla Phe 135 140 145 gtc gtt gtc cac agg atc caa caa att ccc aat gtt cgaaga aga aca 595 Val Val Val His Arg Ile Gln Gln Ile Pro Asn Val Arg ArgArg Thr 150 155 160 att tat gaa tac cac cga gta gag cta caa atg tca aaaatt acc aac 643 Ile Tyr Glu Tyr His Arg Val Glu Leu Gln Met Ser Lys IleThr Asn 165 170 175 att tcg gct gtg gag atg acc cca tta ccc aca tgc ctccag ttt aac 691 Ile Ser Ala Val Glu Met Thr Pro Leu Pro Thr Cys Leu GlnPhe Asn 180 185 190 195 aga tgt ggc ccc tgt gta tct tct cag att ggc ttcaac tgc agt tgg 739 Arg Cys Gly Pro Cys Val Ser Ser Gln Ile Gly Phe AsnCys Ser Trp 200 205 210 tgt agt aaa ctt caa aga tgt tcc agt gga ttt gatcgt cat cgg cag 787 Cys Ser Lys Leu Gln Arg Cys Ser Ser Gly Phe Asp ArgHis Arg Gln 215 220 225 gac tgg gtg gac agt gga tgc cct gaa gag tca aaagag aag atg tgt 835 Asp Trp Val Asp Ser Gly Cys Pro Glu Glu Ser Lys GluLys Met Cys 230 235 240 gag aat aca gaa cca gtg gaa act tct tct cga accacc aca acc ata 883 Glu Asn Thr Glu Pro Val Glu Thr Ser Ser Arg Thr ThrThr Thr Ile 245 250 255 gga gcg aca acc acc cag ttc agg gtc cta act accacc aga aga gca 931 Gly Ala Thr Thr Thr Gln Phe Arg Val Leu Thr Thr ThrArg Arg Ala 260 265 270 275 gtg act tct cag ttt ccc acc agc ctc cct acagaa gat gat acc aag 979 Val Thr Ser Gln Phe Pro Thr Ser Leu Pro Thr GluAsp Asp Thr Lys 280 285 290 ata gca cta cat cta aaa gat aat gga gct tctaca gat gac agt gca 1027 Ile Ala Leu His Leu Lys Asp Asn Gly Ala Ser ThrAsp Asp Ser Ala 295 300 305 gct gag aag aaa ggg gga acc ctc cac gct ggcctc atc gtt gga atc 1075 Ala Glu Lys Lys Gly Gly Thr Leu His Ala Gly LeuIle Val Gly Ile 310 315 320 ctc atc ctg gtc ctc att gta gcc aca gcc attctt gtg aca gtc tat 1123 Leu Ile Leu Val Leu Ile Val Ala Thr Ala Ile LeuVal Thr Val Tyr 325 330 335 atg tat cac cac cca aca tca gca gcc agc atcttc ttt att gag aga 1171 Met Tyr His His Pro Thr Ser Ala Ala Ser Ile PhePhe Ile Glu Arg 340 345 350 355 cgc cca agc aga tgg cct gcg atg aag tttaga aga ggc tct gga cat 1219 Arg Pro Ser Arg Trp Pro Ala Met Lys Phe ArgArg Gly Ser Gly His 360 365 370 cct gcc tat gct gaa gtt gaa cca gtt ggagag aaa gaa ggc ttt att 1267 Pro Ala Tyr Ala Glu Val Glu Pro Val Gly GluLys Glu Gly Phe Ile 375 380 385 gta tca gag cag tgc taaaatttctaggacagaac aacaccagta ctggtttaca 1322 Val Ser Glu Gln Cys 390 ggtgttaagactaaaatttt gcctatacct ttaagacaaa caaacaaaca cacacacaaa 1382 caagctctaagctgctgtag cctgaagaag acaagatttc tggacaagct cagcccagga 1442 aacaaagggtaaacaaaaaa ctaaaactta tacaagatac catttacact gaacatagaa 1502 ttccctagtggaatgtcatc tatagttcac tcggaacatc tcccgtggac ttatctgaag 1562 tatgacaagattataatgct tttggcttag gtgcagggtt gcaaagggat cagaaaaaaa 1622 aaatcataataaagctttag ttcatgaggg atcgacacct ttggttcaaa tgttctctga 1682 tgtctcaaagataactgttt tccaaagcct gaaccctttc actcaaaaga gcaatgatga 1742 atgtctcaagattgctaaga aaaacagccc atgcaagagt gagaacaaac acaaaataag 1802 agattttctacattttcaaa acagatgtgt ggcaaaagga tgttgttttt ctggtctaga 1862 tccatctgtaccaacaagtt catcacttta cagaacgaat ctttttatcc gtacaggagg 1922 ttcaaaccatgtctgcctct tcctttgtaa tgaatgacct ttctatgagc tgtgacaaaa 1982 tttccgaacaattagctaag gatttgggaa gagggggtgg caaacggggc tttctgtttt 2042 cctgcctcagcatgaaaaca tctgatttat gctttatgga agccttacct ccaatcccca 2102 actgttaagtcccatgaaac cacagttgct ctgggctgat ggaaacaaaa ggaaacagta 2162 tgaagagttccttaatcatt tttgaaacaa aaatgttaag ggattttaaa catatgatta 2222 tttttaattttatgcctttt cagtactaaa cacccatttc attgctgatt cctgtctaag 2282 aagccattcacgtcagcatg gcgatagaaa gaatgaaaaa accctgctga atcatacagt 2342 aattttctttaaagcacata gtagttacat aaatatatat atataaatat atttttgttt 2402 ataactaacacaaggcagga tcttgtgact ctaagagtgc gttttgtcat caagacaaaa 2462 cagatgcaagatgcatcact gcattacttc catagagttg taaaataatc cttaatatta 2522 gaatatttttctgtcactta gcaaaagtgg ttcagttcat tgccgcgccc atcatgttct 2582 tgactatttgatccactttt tcgtttatgt caaccccttc cctctctggc taaataaagt 2642 ggatgcagaaagctccttaa atggaa 2668 25 392 PRT Homo sapiens 25 Met Glu Lys Asp LysVal Lys Ile His Gly Ile Leu Ser Asn Thr His 1 5 10 15 Arg Gln Ala AlaArg Val Asn Leu Ser Phe Asp Phe Pro Phe Tyr Gly 20 25 30 His Phe Leu ArgGlu Ile Thr Val Ala Thr Gly Gly Phe Ile Tyr Thr 35 40 45 Gly Glu Val ValHis Arg Met Leu Thr Ala Thr Gln Tyr Ile Ala Pro 50 55 60 Leu Met Ala AsnPhe Asp Pro Ser Val Ser Arg Asn Ser Thr Val Arg 65 70 75 80 Tyr Phe AspAsn Gly Thr Ala Leu Val Val Gln Trp Asp His Val His 85 90 95 Leu Gln AspAsn Tyr Asn Leu Gly Ser Phe Thr Phe Gln Ala Thr Leu 100 105 110 Leu MetAsp Gly Arg Ile Ile Phe Gly Tyr Lys Glu Ile Pro Val Leu 115 120 125 ValThr Gln Ile Ser Ser Thr Asn His Pro Val Lys Val Gly Leu Ser 130 135 140Asp Ala Phe Val Val Val His Arg Ile Gln Gln Ile Pro Asn Val Arg 145 150155 160 Arg Arg Thr Ile Tyr Glu Tyr His Arg Val Glu Leu Gln Met Ser Lys165 170 175 Ile Thr Asn Ile Ser Ala Val Glu Met Thr Pro Leu Pro Thr CysLeu 180 185 190 Gln Phe Asn Arg Cys Gly Pro Cys Val Ser Ser Gln Ile GlyPhe Asn 195 200 205 Cys Ser Trp Cys Ser Lys Leu Gln Arg Cys Ser Ser GlyPhe Asp Arg 210 215 220 His Arg Gln Asp Trp Val Asp Ser Gly Cys Pro GluGlu Ser Lys Glu 225 230 235 240 Lys Met Cys Glu Asn Thr Glu Pro Val GluThr Ser Ser Arg Thr Thr 245 250 255 Thr Thr Ile Gly Ala Thr Thr Thr GlnPhe Arg Val Leu Thr Thr Thr 260 265 270 Arg Arg Ala Val Thr Ser Gln PhePro Thr Ser Leu Pro Thr Glu Asp 275 280 285 Asp Thr Lys Ile Ala Leu HisLeu Lys Asp Asn Gly Ala Ser Thr Asp 290 295 300 Asp Ser Ala Ala Glu LysLys Gly Gly Thr Leu His Ala Gly Leu Ile 305 310 315 320 Val Gly Ile LeuIle Leu Val Leu Ile Val Ala Thr Ala Ile Leu Val 325 330 335 Thr Val TyrMet Tyr His His Pro Thr Ser Ala Ala Ser Ile Phe Phe 340 345 350 Ile GluArg Arg Pro Ser Arg Trp Pro Ala Met Lys Phe Arg Arg Gly 355 360 365 SerGly His Pro Ala Tyr Ala Glu Val Glu Pro Val Gly Glu Lys Glu 370 375 380Gly Phe Ile Val Ser Glu Gln Cys 385 390 26 1179 DNA Homo sapiens 26atggaaaaag ataaagtgaa gattcatgga atattgtcca atactcatcg gcaagctgca 60agagtgaatc tgtccttcga ttttccattt tatggccact tcctacgtga aatcactgtg 120gcaaccgggg gtttcatata cactggagaa gtcgtacatc gaatgctaac agccacacag 180tacatagcac ctttaatggc aaatttcgat cccagtgtat ccagaaattc aactgtcaga 240tattttgata atggcacagc acttgtggtc cagtgggacc atgtacatct ccaggataat 300tataacctgg gaagcttcac attccaggca accctgctca tggatggacg aatcatcttt 360ggatacaaag aaattcctgt cttggtcaca cagataagtt caaccaatca tccagtgaaa 420gtcggactgt ccgatgcatt tgtcgttgtc cacaggatcc aacaaattcc caatgttcga 480agaagaacaa tttatgaata ccaccgagta gagctacaaa tgtcaaaaat taccaacatt 540tcggctgtgg agatgacccc attacccaca tgcctccagt ttaacagatg tggcccctgt 600gtatcttctc agattggctt caactgcagt tggtgtagta aacttcaaag atgttccagt 660ggatttgatc gtcatcggca ggactgggtg gacagtggat gccctgaaga gtcaaaagag 720aagatgtgtg agaatacaga accagtggaa acttcttctc gaaccaccac aaccatagga 780gcgacaacca cccagttcag ggtcctaact accaccagaa gagcagtgac ttctcagttt 840cccaccagcc tccctacaga agatgatacc aagatagcac tacatctaaa agataatgga 900gcttctacag atgacagtgc agctgagaag aaagggggaa ccctccacgc tggcctcatc 960gttggaatcc tcatcctggt cctcattgta gccacagcca ttcttgtgac agtctatatg 1020tatcaccacc caacatcagc agccagcatc ttctttattg agagacgccc aagcagatgg 1080cctgcgatga agtttagaag aggctctgga catcctgcct atgctgaagt tgaaccagtt 1140ggagagaaag aaggctttat tgtatcagag cagtgctaa 1179 27 3095 DNA Homo sapiensCDS (123)..(1712) 27 tttcgttccg ggtcctaccg agaccgatcc gcagcgtttggcccggtcgt gcctattgca 60 tcgggagccc ccgagcaccg gcgaaggact ggcggctggggtagggaggt ggcggcggcg 120 gc atg gcg agg ttc ccg aag gcc gac ctg gcc gctgca gga gtt atg 167 Met Ala Arg Phe Pro Lys Ala Asp Leu Ala Ala Ala GlyVal Met 1 5 10 15 tta ctt tgc cac ttc ttc acg gac cag ttt cag ttc gccgat ggg aaa 215 Leu Leu Cys His Phe Phe Thr Asp Gln Phe Gln Phe Ala AspGly Lys 20 25 30 ccc gga gac caa atc ctt gat tgg cag tat gga gtt act caggcc ttc 263 Pro Gly Asp Gln Ile Leu Asp Trp Gln Tyr Gly Val Thr Gln AlaPhe 35 40 45 cct cac aca gag gag gag gtg gaa gtt gat tca cac gcg tac agccac 311 Pro His Thr Glu Glu Glu Val Glu Val Asp Ser His Ala Tyr Ser His50 55 60 agg tgg aaa aga aac ttg gac ttt ctc aag gcg gta gac acg aac cga359 Arg Trp Lys Arg Asn Leu Asp Phe Leu Lys Ala Val Asp Thr Asn Arg 6570 75 gca agc gtc ggc caa gac tct cct gag ccc aga agc ttc aca gac ctg407 Ala Ser Val Gly Gln Asp Ser Pro Glu Pro Arg Ser Phe Thr Asp Leu 8085 90 95 ctg ctg gat gat ggg cag gac aat aac act cag atc gag gag gat aca455 Leu Leu Asp Asp Gly Gln Asp Asn Asn Thr Gln Ile Glu Glu Asp Thr 100105 110 gac cac aat tac tat ata tct cga ata tat ggt cca tct gat tct gcc503 Asp His Asn Tyr Tyr Ile Ser Arg Ile Tyr Gly Pro Ser Asp Ser Ala 115120 125 agc cgg gat tta tgg gtg aac ata gac caa atg gaa aaa gat aaa gtg551 Ser Arg Asp Leu Trp Val Asn Ile Asp Gln Met Glu Lys Asp Lys Val 130135 140 aag att cat gga ata ttg tcc aat act cat cgg caa gct gca aga gtg599 Lys Ile His Gly Ile Leu Ser Asn Thr His Arg Gln Ala Ala Arg Val 145150 155 aat ctg tcc ttc gat ttt cca ttt tat ggc cac ttc cta cgt gaa atc647 Asn Leu Ser Phe Asp Phe Pro Phe Tyr Gly His Phe Leu Arg Glu Ile 160165 170 175 act gtg gca acc ggg ggt ttc ata tac act gga gaa gtc gta catcga 695 Thr Val Ala Thr Gly Gly Phe Ile Tyr Thr Gly Glu Val Val His Arg180 185 190 atg cta aca gcc aca cag tac ata gca cct tta atg gca aat ttcgat 743 Met Leu Thr Ala Thr Gln Tyr Ile Ala Pro Leu Met Ala Asn Phe Asp195 200 205 ccc agt gta tcc aga aat tca act gtc aga tat ttt gat aat ggcaca 791 Pro Ser Val Ser Arg Asn Ser Thr Val Arg Tyr Phe Asp Asn Gly Thr210 215 220 gca ctt gtg gtc cag tgg gac cat gta cat ctc cag gat aat tataac 839 Ala Leu Val Val Gln Trp Asp His Val His Leu Gln Asp Asn Tyr Asn225 230 235 ctg gga agc ttc aca ttc cag gca acc ctg ctc atg gat gga cgaatc 887 Leu Gly Ser Phe Thr Phe Gln Ala Thr Leu Leu Met Asp Gly Arg Ile240 245 250 255 atc ttt gga tac aaa gaa att cct gtc ttg gtc aca cag ataagt tca 935 Ile Phe Gly Tyr Lys Glu Ile Pro Val Leu Val Thr Gln Ile SerSer 260 265 270 acc aat cat cca gtg aaa gtc gga ctg tcc gat gca ttt gtcgtt gtc 983 Thr Asn His Pro Val Lys Val Gly Leu Ser Asp Ala Phe Val ValVal 275 280 285 cac agg atc caa caa att ccc aat gtt cga aga aga aca atttat gaa 1031 His Arg Ile Gln Gln Ile Pro Asn Val Arg Arg Arg Thr Ile TyrGlu 290 295 300 tac cac cga gta gag cta caa atg tca aaa att acc aac atttcg gct 1079 Tyr His Arg Val Glu Leu Gln Met Ser Lys Ile Thr Asn Ile SerAla 305 310 315 gtg gag atg acc cca tta ccc aca tgc ctc cag ttt aac agatgt ggc 1127 Val Glu Met Thr Pro Leu Pro Thr Cys Leu Gln Phe Asn Arg CysGly 320 325 330 335 ccc tgt gta tct tct cag att ggc ttc aac tgc agt tggtgt agt aaa 1175 Pro Cys Val Ser Ser Gln Ile Gly Phe Asn Cys Ser Trp CysSer Lys 340 345 350 ctt caa aga tgt tcc agt gga ttt gat cgt cat cgg caggac tgg gtg 1223 Leu Gln Arg Cys Ser Ser Gly Phe Asp Arg His Arg Gln AspTrp Val 355 360 365 gac agt gga tgc cct gaa gag tca aaa gag aag atg tgtgag aat aca 1271 Asp Ser Gly Cys Pro Glu Glu Ser Lys Glu Lys Met Cys GluAsn Thr 370 375 380 gaa cca gtg gaa act tct tct cga acc acc aca acc atagga gcg aca 1319 Glu Pro Val Glu Thr Ser Ser Arg Thr Thr Thr Thr Ile GlyAla Thr 385 390 395 acc acc cag ttc agg gtc cta act acc acc aga aga gcagtg act tct 1367 Thr Thr Gln Phe Arg Val Leu Thr Thr Thr Arg Arg Ala ValThr Ser 400 405 410 415 cag ttt ccc acc agc ctc cct aca gaa gat gat accaag ata gca cta 1415 Gln Phe Pro Thr Ser Leu Pro Thr Glu Asp Asp Thr LysIle Ala Leu 420 425 430 cat cta aaa gat aat gga gct tct aca gat gac agtgca gct gag aag 1463 His Leu Lys Asp Asn Gly Ala Ser Thr Asp Asp Ser AlaAla Glu Lys 435 440 445 aaa ggg gga acc ctc cac gct ggc ctc atc gtt ggaatc ctc atc ctg 1511 Lys Gly Gly Thr Leu His Ala Gly Leu Ile Val Gly IleLeu Ile Leu 450 455 460 gtc ctc att gta gcc aca gcc att ctt gtg aca gtctat atg tat cac 1559 Val Leu Ile Val Ala Thr Ala Ile Leu Val Thr Val TyrMet Tyr His 465 470 475 cac cca aca tca gca gcc agc atc ttc ttt att gagaga cgc cca agc 1607 His Pro Thr Ser Ala Ala Ser Ile Phe Phe Ile Glu ArgArg Pro Ser 480 485 490 495 aga tgg cct gcg atg aag ttt aga aga ggc tctgga cat cct gcc tat 1655 Arg Trp Pro Ala Met Lys Phe Arg Arg Gly Ser GlyHis Pro Ala Tyr 500 505 510 gct gaa gtt gaa cca gtt gga gag aaa gaa ggcttt att gta tca gag 1703 Ala Glu Val Glu Pro Val Gly Glu Lys Glu Gly PheIle Val Ser Glu 515 520 525 cag tgc taa aatttctagg acagaacaac accagtactggtttacaggt 1752 Gln Cys gttaagacta aaattttgcc tataccttta agacaaacaaacaaacacac acacaaacaa 1812 gctctaagct gctgtagcct gaagaagaca agatttctggacaagctcag cccaggaaac 1872 aaagggtaaa caaaaaacta aaacttatac aagataccatttacactgaa catagaattc 1932 cctagtggaa tgtcatctat agttcactcg gaacatctcccgtggactta tctgaagtat 1992 gacaagatta taatgctttt ggcttaggtg cagggttgcaaagggatcag aaaaaaaaaa 2052 tcataataaa gctttagttc atgagggatc gacacctttggttcaaatgt tctctgatgt 2112 ctcaaagata actgttttcc aaagcctgaa ccctttcactcaaaagagca atgatgaatg 2172 tctcaagatt gctaagaaaa acagcccatg caagagtgagaacaaacaca aaataagaga 2232 ttttctacat tttcaaaaca gatgtgtggc aaaaggatgttgtttttctg gtctagatcc 2292 atctgtacca acaagttcat cactttacag aacgaatctttttatccgta caggaggttc 2352 aaaccatgtc tgcctcttcc tttgtaatga atgacctttctatgagctgt gacaaaattt 2412 ccgaacaatt agctaaggat ttgggaagag ggggtggcaaacggggcttt ctgttttcct 2472 gcctcagcat gaaaacatct gatttatgct ttatggaagccttacctcca atccccaact 2532 gttaagtccc atgaaaccac agttgctctg ggctgatggaaacaaaagga aacagtatga 2592 agagttcctt aatcattttt gaaacaaaaa tgttaagggattttaaacat atgattattt 2652 ttaattttat gccttttcag tactaaacac ccatttcattgctgattcct gtctaagaag 2712 ccattcacgt cagcatggcg atagaaagaa tgaaaaaaccctgctgaatc atacagtaat 2772 tttctttaaa gcacatagta gttacataaa tatatatatataaatatatt tttgtttata 2832 actaacacaa ggcaggatct tgtgactcta agagtgcgttttgtcatcaa gacaaaacag 2892 atgcaagatg catcactgca ttacttccat agagttgtaaaataatcctt aatattagaa 2952 tatttttctg tcacttagca aaagtggttc agttcattgccgcgcccatc atgttcttga 3012 ctatttgatc cactttttcg tttatgtcaa ccccttccctctctggctaa ataaagtgga 3072 tgcagaaagc tccttaaatg gaa 3095 28 529 PRTHomo sapiens 28 Met Ala Arg Phe Pro Lys Ala Asp Leu Ala Ala Ala Gly ValMet Leu 1 5 10 15 Leu Cys His Phe Phe Thr Asp Gln Phe Gln Phe Ala AspGly Lys Pro 20 25 30 Gly Asp Gln Ile Leu Asp Trp Gln Tyr Gly Val Thr GlnAla Phe Pro 35 40 45 His Thr Glu Glu Glu Val Glu Val Asp Ser His Ala TyrSer His Arg 50 55 60 Trp Lys Arg Asn Leu Asp Phe Leu Lys Ala Val Asp ThrAsn Arg Ala 65 70 75 80 Ser Val Gly Gln Asp Ser Pro Glu Pro Arg Ser PheThr Asp Leu Leu 85 90 95 Leu Asp Asp Gly Gln Asp Asn Asn Thr Gln Ile GluGlu Asp Thr Asp 100 105 110 His Asn Tyr Tyr Ile Ser Arg Ile Tyr Gly ProSer Asp Ser Ala Ser 115 120 125 Arg Asp Leu Trp Val Asn Ile Asp Gln MetGlu Lys Asp Lys Val Lys 130 135 140 Ile His Gly Ile Leu Ser Asn Thr HisArg Gln Ala Ala Arg Val Asn 145 150 155 160 Leu Ser Phe Asp Phe Pro PheTyr Gly His Phe Leu Arg Glu Ile Thr 165 170 175 Val Ala Thr Gly Gly PheIle Tyr Thr Gly Glu Val Val His Arg Met 180 185 190 Leu Thr Ala Thr GlnTyr Ile Ala Pro Leu Met Ala Asn Phe Asp Pro 195 200 205 Ser Val Ser ArgAsn Ser Thr Val Arg Tyr Phe Asp Asn Gly Thr Ala 210 215 220 Leu Val ValGln Trp Asp His Val His Leu Gln Asp Asn Tyr Asn Leu 225 230 235 240 GlySer Phe Thr Phe Gln Ala Thr Leu Leu Met Asp Gly Arg Ile Ile 245 250 255Phe Gly Tyr Lys Glu Ile Pro Val Leu Val Thr Gln Ile Ser Ser Thr 260 265270 Asn His Pro Val Lys Val Gly Leu Ser Asp Ala Phe Val Val Val His 275280 285 Arg Ile Gln Gln Ile Pro Asn Val Arg Arg Arg Thr Ile Tyr Glu Tyr290 295 300 His Arg Val Glu Leu Gln Met Ser Lys Ile Thr Asn Ile Ser AlaVal 305 310 315 320 Glu Met Thr Pro Leu Pro Thr Cys Leu Gln Phe Asn ArgCys Gly Pro 325 330 335 Cys Val Ser Ser Gln Ile Gly Phe Asn Cys Ser TrpCys Ser Lys Leu 340 345 350 Gln Arg Cys Ser Ser Gly Phe Asp Arg His ArgGln Asp Trp Val Asp 355 360 365 Ser Gly Cys Pro Glu Glu Ser Lys Glu LysMet Cys Glu Asn Thr Glu 370 375 380 Pro Val Glu Thr Ser Ser Arg Thr ThrThr Thr Ile Gly Ala Thr Thr 385 390 395 400 Thr Gln Phe Arg Val Leu ThrThr Thr Arg Arg Ala Val Thr Ser Gln 405 410 415 Phe Pro Thr Ser Leu ProThr Glu Asp Asp Thr Lys Ile Ala Leu His 420 425 430 Leu Lys Asp Asn GlyAla Ser Thr Asp Asp Ser Ala Ala Glu Lys Lys 435 440 445 Gly Gly Thr LeuHis Ala Gly Leu Ile Val Gly Ile Leu Ile Leu Val 450 455 460 Leu Ile ValAla Thr Ala Ile Leu Val Thr Val Tyr Met Tyr His His 465 470 475 480 ProThr Ser Ala Ala Ser Ile Phe Phe Ile Glu Arg Arg Pro Ser Arg 485 490 495Trp Pro Ala Met Lys Phe Arg Arg Gly Ser Gly His Pro Ala Tyr Ala 500 505510 Glu Val Glu Pro Val Gly Glu Lys Glu Gly Phe Ile Val Ser Glu Gln 515520 525 Cys 29 1590 DNA Homo sapiens 29 atggcgaggt tcccgaaggc cgacctggccgctgcaggag ttatgttact ttgccacttc 60 ttcacggacc agtttcagtt cgccgatgggaaacccggag accaaatcct tgattggcag 120 tatggagtta ctcaggcctt ccctcacacagaggaggagg tggaagttga ttcacacgcg 180 tacagccaca ggtggaaaag aaacttggactttctcaagg cggtagacac gaaccgagca 240 agcgtcggcc aagactctcc tgagcccagaagcttcacag acctgctgct ggatgatggg 300 caggacaata acactcagat cgaggaggatacagaccaca attactatat atctcgaata 360 tatggtccat ctgattctgc cagccgggatttatgggtga acatagacca aatggaaaaa 420 gataaagtga agattcatgg aatattgtccaatactcatc ggcaagctgc aagagtgaat 480 ctgtccttcg attttccatt ttatggccacttcctacgtg aaatcactgt ggcaaccggg 540 ggtttcatat acactggaga agtcgtacatcgaatgctaa cagccacaca gtacatagca 600 cctttaatgg caaatttcga tcccagtgtatccagaaatt caactgtcag atattttgat 660 aatggcacag cacttgtggt ccagtgggaccatgtacatc tccaggataa ttataacctg 720 ggaagcttca cattccaggc aaccctgctcatggatggac gaatcatctt tggatacaaa 780 gaaattcctg tcttggtcac acagataagttcaaccaatc atccagtgaa agtcggactg 840 tccgatgcat ttgtcgttgt ccacaggatccaacaaattc ccaatgttcg aagaagaaca 900 atttatgaat accaccgagt agagctacaaatgtcaaaaa ttaccaacat ttcggctgtg 960 gagatgaccc cattacccac atgcctccagtttaacagat gtggcccctg tgtatcttct 1020 cagattggct tcaactgcag ttggtgtagtaaacttcaaa gatgttccag tggatttgat 1080 cgtcatcggc aggactgggt ggacagtggatgccctgaag agtcaaaaga gaagatgtgt 1140 gagaatacag aaccagtgga aacttcttctcgaaccacca caaccatagg agcgacaacc 1200 acccagttca gggtcctaac taccaccagaagagcagtga cttctcagtt tcccaccagc 1260 ctccctacag aagatgatac caagatagcactacatctaa aagataatgg agcttctaca 1320 gatgacagtg cagctgagaa gaaagggggaaccctccacg ctggcctcat cgttggaatc 1380 ctcatcctgg tcctcattgt agccacagccattcttgtga cagtctatat gtatcaccac 1440 ccaacatcag cagccagcat cttctttattgagagacgcc caagcagatg gcctgcgatg 1500 aagtttagaa gaggctctgg acatcctgcctatgctgaag ttgaaccagt tggagagaaa 1560 gaaggcttta ttgtatcaga gcagtgctaa1590 30 30 PRT Homo sapiens 30 Met Ala Arg Phe Pro Lys Ala Asp Leu AlaAla Ala Gly Val Met Leu 1 5 10 15 Leu Cys His Phe Phe Thr Asp Gln PheGln Phe Ala Asp Gly 20 25 30 31 499 PRT Homo sapiens 31 Lys Pro Gly AspGln Ile Leu Asp Trp Gln Tyr Gly Val Thr Gln Ala 1 5 10 15 Phe Pro HisThr Glu Glu Glu Val Glu Val Asp Ser His Ala Tyr Ser 20 25 30 His Arg TrpLys Arg Asn Leu Asp Phe Leu Lys Ala Val Asp Thr Asn 35 40 45 Arg Ala SerVal Gly Gln Asp Ser Pro Glu Pro Arg Ser Phe Thr Asp 50 55 60 Leu Leu LeuAsp Asp Gly Gln Asp Asn Asn Thr Gln Ile Glu Glu Asp 65 70 75 80 Thr AspHis Asn Tyr Tyr Ile Ser Arg Ile Tyr Gly Pro Ser Asp Ser 85 90 95 Ala SerArg Asp Leu Trp Val Asn Ile Asp Gln Met Glu Lys Asp Lys 100 105 110 ValLys Ile His Gly Ile Leu Ser Asn Thr His Arg Gln Ala Ala Arg 115 120 125Val Asn Leu Ser Phe Asp Phe Pro Phe Tyr Gly His Phe Leu Arg Glu 130 135140 Ile Thr Val Ala Thr Gly Gly Phe Ile Tyr Thr Gly Glu Val Val His 145150 155 160 Arg Met Leu Thr Ala Thr Gln Tyr Ile Ala Pro Leu Met Ala AsnPhe 165 170 175 Asp Pro Ser Val Ser Arg Asn Ser Thr Val Arg Tyr Phe AspAsn Gly 180 185 190 Thr Ala Leu Val Val Gln Trp Asp His Val His Leu GlnAsp Asn Tyr 195 200 205 Asn Leu Gly Ser Phe Thr Phe Gln Ala Thr Leu LeuMet Asp Gly Arg 210 215 220 Ile Ile Phe Gly Tyr Lys Glu Ile Pro Val LeuVal Thr Gln Ile Ser 225 230 235 240 Ser Thr Asn His Pro Val Lys Val GlyLeu Ser Asp Ala Phe Val Val 245 250 255 Val His Arg Ile Gln Gln Ile ProAsn Val Arg Arg Arg Thr Ile Tyr 260 265 270 Glu Tyr His Arg Val Glu LeuGln Met Ser Lys Ile Thr Asn Ile Ser 275 280 285 Ala Val Glu Met Thr ProLeu Pro Thr Cys Leu Gln Phe Asn Arg Cys 290 295 300 Gly Pro Cys Val SerSer Gln Ile Gly Phe Asn Cys Ser Trp Cys Ser 305 310 315 320 Lys Leu GlnArg Cys Ser Ser Gly Phe Asp Arg His Arg Gln Asp Trp 325 330 335 Val AspSer Gly Cys Pro Glu Glu Ser Lys Glu Lys Met Cys Glu Asn 340 345 350 ThrGlu Pro Val Glu Thr Ser Ser Arg Thr Thr Thr Thr Ile Gly Ala 355 360 365Thr Thr Thr Gln Phe Arg Val Leu Thr Thr Thr Arg Arg Ala Val Thr 370 375380 Ser Gln Phe Pro Thr Ser Leu Pro Thr Glu Asp Asp Thr Lys Ile Ala 385390 395 400 Leu His Leu Lys Asp Asn Gly Ala Ser Thr Asp Asp Ser Ala AlaGlu 405 410 415 Lys Lys Gly Gly Thr Leu His Ala Gly Leu Ile Val Gly IleLeu Ile 420 425 430 Leu Val Leu Ile Val Ala Thr Ala Ile Leu Val Thr ValTyr Met Tyr 435 440 445 His His Pro Thr Ser Ala Ala Ser Ile Phe Phe IleGlu Arg Arg Pro 450 455 460 Ser Arg Trp Pro Ala Met Lys Phe Arg Arg GlySer Gly His Pro Ala 465 470 475 480 Tyr Ala Glu Val Glu Pro Val Gly GluLys Glu Gly Phe Ile Val Ser 485 490 495 Glu Gln Cys 32 28 PRT Homosapiens 32 Leu His Ala Gly Leu Ile Val Gly Ile Leu Ile Leu Val Leu IleVal 1 5 10 15 Ala Thr Ala Ile Leu Val Thr Val Tyr Met Tyr His 20 25 331351 DNA Homo sapiens CDS (5)..(1351) 33 cggc atg gcg agg ttc ccg aaggcc gac ctg gcc gct gca gga gtt atg 49 Met Ala Arg Phe Pro Lys Ala AspLeu Ala Ala Ala Gly Val Met 1 5 10 15 tta ctt tgc cac ttc ttc acg gaccag ttt cag ttc gcc gat ggg aaa 97 Leu Leu Cys His Phe Phe Thr Asp GlnPhe Gln Phe Ala Asp Gly Lys 20 25 30 ccc gga gac caa atc ctt gat tgg cagtat gga gtt act cag gcc ttc 145 Pro Gly Asp Gln Ile Leu Asp Trp Gln TyrGly Val Thr Gln Ala Phe 35 40 45 cct cac aca gag gag gag gtg gaa gtt gattca cac gcg tac agc cac 193 Pro His Thr Glu Glu Glu Val Glu Val Asp SerHis Ala Tyr Ser His 50 55 60 agg tgg aaa aga aac ttg gac ttt ctc aag gcggta gac acg aac cga 241 Arg Trp Lys Arg Asn Leu Asp Phe Leu Lys Ala ValAsp Thr Asn Arg 65 70 75 gca agc gtc ggc caa gac tct cct gag ccc aga agcttc aca gac ctg 289 Ala Ser Val Gly Gln Asp Ser Pro Glu Pro Arg Ser PheThr Asp Leu 80 85 90 95 ctg ctg gat gat ggg cag gac aat aac act cag atcgag gag gat aca 337 Leu Leu Asp Asp Gly Gln Asp Asn Asn Thr Gln Ile GluGlu Asp Thr 100 105 110 gac cac aat tac tat ata tct cga ata tat ggt ccatct gat tct gcc 385 Asp His Asn Tyr Tyr Ile Ser Arg Ile Tyr Gly Pro SerAsp Ser Ala 115 120 125 agc cgg gat tta tgg gtg aac ata gac caa atg gaaaaa gat aaa gtg 433 Ser Arg Asp Leu Trp Val Asn Ile Asp Gln Met Glu LysAsp Lys Val 130 135 140 aag att cat gga ata ttg tcc aat act cat cgg caagct gca aga gtg 481 Lys Ile His Gly Ile Leu Ser Asn Thr His Arg Gln AlaAla Arg Val 145 150 155 aat ctg tcc ttc gat ttt cca ttt tat ggc cac ttccta cgt gaa atc 529 Asn Leu Ser Phe Asp Phe Pro Phe Tyr Gly His Phe LeuArg Glu Ile 160 165 170 175 act gtg gca acc ggg ggt ttc ata tac act ggagaa gtc gta cat cga 577 Thr Val Ala Thr Gly Gly Phe Ile Tyr Thr Gly GluVal Val His Arg 180 185 190 atg cta aca gcc aca cag tac ata gca cct ttaatg gca aat ttc gat 625 Met Leu Thr Ala Thr Gln Tyr Ile Ala Pro Leu MetAla Asn Phe Asp 195 200 205 ccc agt gta tcc aga aat tca act gtc aga tatttt gat aat ggc aca 673 Pro Ser Val Ser Arg Asn Ser Thr Val Arg Tyr PheAsp Asn Gly Thr 210 215 220 gca ctt gtg gtc cag tgg gac cat gta cat ctccag gat aat tat aac 721 Ala Leu Val Val Gln Trp Asp His Val His Leu GlnAsp Asn Tyr Asn 225 230 235 ctg gga agc ttc aca ttc cag gca acc ctg ctcatg gat gga cga atc 769 Leu Gly Ser Phe Thr Phe Gln Ala Thr Leu Leu MetAsp Gly Arg Ile 240 245 250 255 atc ttt gga tac aaa gaa att cct gtc ttggtc aca cag ata agt tca 817 Ile Phe Gly Tyr Lys Glu Ile Pro Val Leu ValThr Gln Ile Ser Ser 260 265 270 acc aat cat cca gtg aaa gtc gga ctg tccgat gca ttt gtc gtt gtc 865 Thr Asn His Pro Val Lys Val Gly Leu Ser AspAla Phe Val Val Val 275 280 285 cac agg atc caa caa att ccc aat gtt cgaaga aga aca att tat gaa 913 His Arg Ile Gln Gln Ile Pro Asn Val Arg ArgArg Thr Ile Tyr Glu 290 295 300 tac cac cga gta gag cta caa atg tca aaaatt acc aac att tcg gct 961 Tyr His Arg Val Glu Leu Gln Met Ser Lys IleThr Asn Ile Ser Ala 305 310 315 gtg gag atg acc cca tta ccc aca tgc ctccag ttt aac aga tgt ggc 1009 Val Glu Met Thr Pro Leu Pro Thr Cys Leu GlnPhe Asn Arg Cys Gly 320 325 330 335 ccc tgt gta tct tct cag att ggc ttcaac tgc agt tgg tgt agt aaa 1057 Pro Cys Val Ser Ser Gln Ile Gly Phe AsnCys Ser Trp Cys Ser Lys 340 345 350 ctt caa aga tgt tcc agt gga ttt gatcgt cat cgg cag gac tgg gtg 1105 Leu Gln Arg Cys Ser Ser Gly Phe Asp ArgHis Arg Gln Asp Trp Val 355 360 365 gac agt gga tgc cct gaa gag tca aaagag aag atg tgt gag aat aca 1153 Asp Ser Gly Cys Pro Glu Glu Ser Lys GluLys Met Cys Glu Asn Thr 370 375 380 gaa cca gtg gaa act tct tct cga accacc aca acc ata gga gcg aca 1201 Glu Pro Val Glu Thr Ser Ser Arg Thr ThrThr Thr Ile Gly Ala Thr 385 390 395 acc acc cag ttc agg gtc cta act accacc aga aga gca gtg act tct 1249 Thr Thr Gln Phe Arg Val Leu Thr Thr ThrArg Arg Ala Val Thr Ser 400 405 410 415 cag ttt ccc acc agc ctc cct acagaa gat gat acc aag ata gca cta 1297 Gln Phe Pro Thr Ser Leu Pro Thr GluAsp Asp Thr Lys Ile Ala Leu 420 425 430 cat cta aaa gat aat gga gct tctaca gat gac agt gca gct gag aag 1345 His Leu Lys Asp Asn Gly Ala Ser ThrAsp Asp Ser Ala Ala Glu Lys 435 440 445 aaa ggg 1351 Lys Gly 34 449 PRTHomo sapiens 34 Met Ala Arg Phe Pro Lys Ala Asp Leu Ala Ala Ala Gly ValMet Leu 1 5 10 15 Leu Cys His Phe Phe Thr Asp Gln Phe Gln Phe Ala AspGly Lys Pro 20 25 30 Gly Asp Gln Ile Leu Asp Trp Gln Tyr Gly Val Thr GlnAla Phe Pro 35 40 45 His Thr Glu Glu Glu Val Glu Val Asp Ser His Ala TyrSer His Arg 50 55 60 Trp Lys Arg Asn Leu Asp Phe Leu Lys Ala Val Asp ThrAsn Arg Ala 65 70 75 80 Ser Val Gly Gln Asp Ser Pro Glu Pro Arg Ser PheThr Asp Leu Leu 85 90 95 Leu Asp Asp Gly Gln Asp Asn Asn Thr Gln Ile GluGlu Asp Thr Asp 100 105 110 His Asn Tyr Tyr Ile Ser Arg Ile Tyr Gly ProSer Asp Ser Ala Ser 115 120 125 Arg Asp Leu Trp Val Asn Ile Asp Gln MetGlu Lys Asp Lys Val Lys 130 135 140 Ile His Gly Ile Leu Ser Asn Thr HisArg Gln Ala Ala Arg Val Asn 145 150 155 160 Leu Ser Phe Asp Phe Pro PheTyr Gly His Phe Leu Arg Glu Ile Thr 165 170 175 Val Ala Thr Gly Gly PheIle Tyr Thr Gly Glu Val Val His Arg Met 180 185 190 Leu Thr Ala Thr GlnTyr Ile Ala Pro Leu Met Ala Asn Phe Asp Pro 195 200 205 Ser Val Ser ArgAsn Ser Thr Val Arg Tyr Phe Asp Asn Gly Thr Ala 210 215 220 Leu Val ValGln Trp Asp His Val His Leu Gln Asp Asn Tyr Asn Leu 225 230 235 240 GlySer Phe Thr Phe Gln Ala Thr Leu Leu Met Asp Gly Arg Ile Ile 245 250 255Phe Gly Tyr Lys Glu Ile Pro Val Leu Val Thr Gln Ile Ser Ser Thr 260 265270 Asn His Pro Val Lys Val Gly Leu Ser Asp Ala Phe Val Val Val His 275280 285 Arg Ile Gln Gln Ile Pro Asn Val Arg Arg Arg Thr Ile Tyr Glu Tyr290 295 300 His Arg Val Glu Leu Gln Met Ser Lys Ile Thr Asn Ile Ser AlaVal 305 310 315 320 Glu Met Thr Pro Leu Pro Thr Cys Leu Gln Phe Asn ArgCys Gly Pro 325 330 335 Cys Val Ser Ser Gln Ile Gly Phe Asn Cys Ser TrpCys Ser Lys Leu 340 345 350 Gln Arg Cys Ser Ser Gly Phe Asp Arg His ArgGln Asp Trp Val Asp 355 360 365 Ser Gly Cys Pro Glu Glu Ser Lys Glu LysMet Cys Glu Asn Thr Glu 370 375 380 Pro Val Glu Thr Ser Ser Arg Thr ThrThr Thr Ile Gly Ala Thr Thr 385 390 395 400 Thr Gln Phe Arg Val Leu ThrThr Thr Arg Arg Ala Val Thr Ser Gln 405 410 415 Phe Pro Thr Ser Leu ProThr Glu Asp Asp Thr Lys Ile Ala Leu His 420 425 430 Leu Lys Asp Asn GlyAla Ser Thr Asp Asp Ser Ala Ala Glu Lys Lys 435 440 445 Gly 35 425 PRTHomo sapiens 35 Pro Arg Val Arg Pro Arg Val Arg Thr Asp His Asn Tyr TyrIle Ser 1 5 10 15 Arg Ile Tyr Gly Pro Ser Asp Ser Ala Ser Arg Asp LeuTrp Val Asn 20 25 30 Ile Asp Gln Met Glu Lys Asp Lys Val Lys Ile His GlyIle Leu Ser 35 40 45 Asn Thr His Arg Gln Ala Ala Arg Val Asn Leu Ser PheAsp Phe Pro 50 55 60 Phe Tyr Gly His Phe Leu Arg Glu Ile Thr Val Ala ThrGly Gly Phe 65 70 75 80 Ile Tyr Thr Gly Glu Val Val His Arg Met Leu ThrAla Thr Gln Tyr 85 90 95 Ile Ala Pro Leu Met Ala Asn Phe Asp Pro Ser ValSer Arg Asn Ser 100 105 110 Thr Val Arg Tyr Phe Asp Asn Gly Thr Ala LeuVal Val Gln Trp Asp 115 120 125 His Val His Leu Gln Asp Asn Tyr Asn LeuGly Ser Phe Thr Phe Gln 130 135 140 Ala Thr Leu Leu Met Asp Gly Arg IleIle Phe Gly Tyr Lys Glu Ile 145 150 155 160 Pro Val Leu Val Thr Gln IleSer Ser Thr Asn His Pro Val Lys Val 165 170 175 Gly Leu Ser Asp Ala PheVal Val Val His Arg Ile Gln Gln Ile Pro 180 185 190 Asn Val Arg Arg ArgThr Ile Tyr Glu Tyr His Arg Val Glu Leu Gln 195 200 205 Met Ser Lys IleThr Asn Ile Ser Ala Val Glu Met Thr Pro Leu Pro 210 215 220 Thr Cys LeuGln Phe Asn Arg Cys Gly Pro Cys Val Ser Ser Gln Ile 225 230 235 240 GlyPhe Asn Cys Ser Trp Cys Ser Lys Leu Gln Arg Cys Ser Ser Gly 245 250 255Phe Asp Arg His Arg Gln Asp Trp Val Asp Ser Gly Cys Pro Glu Glu 260 265270 Ser Lys Glu Lys Met Cys Glu Asn Thr Glu Pro Val Glu Thr Phe Leu 275280 285 Glu Pro Pro Gln Pro Glu Arg Gln Pro Pro Ser Ser Gly Ser Leu Pro290 295 300 Pro Glu Asp Ala Val Thr Ser Gln Phe Pro Thr Ser Leu Pro ThrGlu 305 310 315 320 Asp Asp Thr Lys Ile Ala Leu His Leu Lys Asp Asn GlyAla Ser Thr 325 330 335 Asp Asp Ser Ala Ala Glu Lys Lys Gly Gly Thr LeuHis Ala Gly Leu 340 345 350 Ile Val Gly Ile Leu Ile Leu Val Leu Ile ValAla Thr Ala Ile Leu 355 360 365 Val Thr Val Tyr Met Tyr His His Pro ThrSer Ala Ala Ser Ile Phe 370 375 380 Phe Ile Glu Arg Arg Pro Ser Arg TrpPro Ala Met Lys Phe Arg Arg 385 390 395 400 Gly Ser Gly His Pro Ala TyrAla Glu Val Glu Pro Val Gly Glu Lys 405 410 415 Glu Gly Phe Ile Val SerGlu Gln Cys 420 425 36 431 PRT Homo sapiens 36 Leu Ala Met Asp Thr LeuPro Asp Asn Arg Thr Arg Val Val Glu Asp 1 5 10 15 Asn His Ser Tyr TyrVal Ser Arg Leu Tyr Gly Pro Ser Glu Pro His 20 25 30 Ser Arg Glu Leu TrpVal Asp Val Ala Glu Ala Asn Arg Ser Gln Val 35 40 45 Lys Ile His Thr IleLeu Ser Asn Thr His Arg Gln Ala Ser Arg Val 50 55 60 Val Leu Ser Phe AspPhe Pro Phe Tyr Gly His Pro Leu Arg Gln Ile 65 70 75 80 Thr Ile Ala ThrGly Gly Phe Ile Phe Met Gly Asp Val Ile His Arg 85 90 95 Met Leu Thr AlaThr Gln Tyr Val Ala Pro Leu Met Ala Asn Phe Asn 100 105 110 Pro Gly TyrSer Asp Asn Ser Thr Val Val Tyr Phe Asp Asn Gly Thr 115 120 125 Val PheVal Val Gln Trp Asp His Val Tyr Leu Gln Gly Trp Glu Asp 130 135 140 LysGly Ser Phe Thr Phe Gln Ala Ala Leu His His Asp Gly Arg Ile 145 150 155160 Val Phe Ala Tyr Lys Glu Ile Pro Met Ser Val Pro Glu Ile Ser Ser 165170 175 Ser Gln His Pro Val Lys Thr Gly Leu Ser Asp Ala Phe Met Ile Leu180 185 190 Asn Pro Ser Pro Asp Val Pro Glu Ser Arg Arg Arg Ser Ile PheGlu 195 200 205 Tyr His Arg Ile Glu Leu Asp Pro Ser Lys Val Thr Ser MetSer Ala 210 215 220 Val Glu Phe Thr Pro Leu Pro Thr Cys Leu Gln His ArgSer Cys Asp 225 230 235 240 Ala Cys Met Ser Ser Asp Leu Thr Phe Asn CysSer Trp Cys His Val 245 250 255 Leu Gln Arg Cys Ser Ser Gly Phe Asp ArgTyr Arg Gln Glu Trp Asp 260 265 270 Gly Thr Met Gly Cys Ala Gln Glu AlaGlu Gly Gln Asp Val Arg Gly 275 280 285 Leu Pro Gly Met Arg Thr Thr ThrSer Ala Ser Pro Asp Thr Ser Phe 290 295 300 Ser Pro Tyr Asp Gly Asp LeuThr Thr Thr Ser Ser Ser Leu Phe Ile 305 310 315 320 Asp Ser Leu Thr ThrGlu Asp Asp Thr Lys Leu Asn Pro Tyr Ala Gly 325 330 335 Gly Asp Gly LeuGln Asn Asn Leu Ser Pro Lys Thr Lys Gly Thr Pro 340 345 350 Val His LeuGly Thr Ile Val Gly Ile Val Leu Ala Val Leu Leu Val 355 360 365 Ala AlaIle Ile Leu Ala Gly Ile Tyr Ile Asn Gly His Pro Thr Ser 370 375 380 AsnAla Ala Leu Phe Phe Ile Glu Arg Arg Pro His His Trp Pro Ala 385 390 395400 Met Lys Phe Arg Ser His Pro Asp His Ser Thr Tyr Ala Glu Val Glu 405410 415 Pro Ser Gly His Glu Lys Glu Gly Phe Met Glu Ala Glu Gln Cys 420425 430

We claim:
 1. An isolated polynucleotide comprising a nucleotide sequenceselected from the group consisting of SEQ ID NO: 1-21, 24, 26-27, 29, or33, the translated protein coding portion thereof, the mature proteincoding portion thereof the extracellular portion thereof, or the activedomain thereof.
 2. An isolated polynucleotide encoding a polypeptidewith biological activity, which polynucleotide hybridizes to thecomplement of a polynucleotide of claim 1 under stringent hybridizationconditions.
 3. An isolated polynucleotide encoding a polypeptide withbiological activity, said polynucleotide having greater than about 90%sequence identity with the polynucleotide of claim
 1. 4. Thepolynucleotide of claim 1 which is a DNA sequence.
 5. An isolatedpolynucleotide which comprises the complement of the polynucleotide ofclaim
 1. 6. A vector comprising the polynucleotide of claim
 1. 7. Anexpression vector comprising the polynucleotide of claim
 1. 8. A hostcell genetically engineered to express the polynucleotide of claim
 1. 9.The host cell of claim 8 wherein the polynucleotide is in operativeassociation with a regulatory sequence that controls expression of thepolynucleotide in the host cell.
 10. An isolated polypeptide comprisingan amino acid sequence which is at least 80% identical to the amino acidsequence selected from the group consisting of SEQ ID NO: 25, 28, 30-32,34, or 35, the translated protein coding portion thereof, the matureprotein coding portion thereof, the extracellular portion thereof, orthe active domain thereof.
 11. A composition comprising the polypeptideof claim 10 and a carrier.
 12. A polypeptide, having stem cell growthfactor-like activity comprising at least ten consecutive amino acidsfrom the polypeptide sequences selected from the group consisting of SEQID NO: 25, 28, 30-32, 34, or
 35. 13. The polypeptide of claim 12comprising at least five consecutive amino acids from the polypeptidesequences selected from the group consisting of SEQ ID NO: 25, 28,30-32, 34, or
 35. 14. A polynucleotide encoding a polypeptide accordingto claim
 12. 15. A polynucleotide encoding a polypeptide according toclaim
 13. 16. A polynucleotide encoding a polypeptide according to claim10.
 17. An antibody specific for the polypeptide of claim
 10. 18. Amethod for detecting the polynucleotide of claim 1 in a sample.comprising: a) contacting the sample with a compound that binds to andforms a complex with the polynucleotide of claim 1 for a periodsufficient to form the complex; and b) detecting the complex, so that ifa complex is detected, the polynucleotide of claim 1 is detected.
 19. Amethod for detecting the polynucleotide of claim 1 in a sample,comprising: a) contacting the sample under stringent hybridizationconditions with nucleic acid primers that anneal to the polynucleotideof claim 1 under such conditions; b) amplifying a product comprising atleast a portion of the polynucleotide of claim 1; and c) detecting saidproduct and thereby the polynucleotide of claim 1 in the sample.
 20. Themethod of claim 19, wherein the polynucleotide comprises an RNA moleculeand the method further comprises reverse transcribing an annealed RNAmolecule into a cDNA polynucleotide.
 21. A method for detecting thepolypeptide of claim 10 in a sample. comprising: a) contacting thesample with a compound that binds to and forms a complex with thepolypeptide under conditions and for a period sufficient to form thecomplex; and b) detecting formation of the complex, so that if a complexformation is detected, the polypeptide of claim 10 is detected.
 22. Amethod for identifying a compound that binds to the polypeptide of claim10, comprising: a) contacting the compound with the polypeptide of claim10 under conditions and for a time sufficient to form apolypeptide/compound complex; and b) detecting the complex, so that ifthe polypeptide/compound complex is detected, a compound that binds to,the polypeptide of claim 10 is identified.
 23. A method for identifyinga compound that binds to the polypeptide of claim 10 comprising: a)contacting the compound with the polypeptide of claim 10, in a cell, fora time sufficient to form a polypeptide/compound complex, wherein thecomplex drives expression of a reporter gene sequence in the cell; andb) detecting the complex by detecting reporter gene sequence expression,so that if the polypeptide/compound complex is detected, a compound thatbinds to the polypeptide of claim 10 is identified.
 24. A method ofproducing a stem cell growth factor-like polypeptide, comprising. a)culturing the host cell of claim 8 under conditions sufficient toexpress the polypeptide in said cell; and b) isolating the polypeptidefrom the cell culture or cells of step (a).
 25. A kit comprising thepolypeptide of claim
 10. 26. A nucleic acid array comprising thepolynucleotide of claim 1 or a unique segment of the polynucleotide ofclaim 1 attached to a surface.
 27. The array of claim 26, wherein thearray detects full-matches to the polynucleotide or a unique segment ofthe polynucleotide of claim
 1. 28. The array of claim 26, wherein thearray detects mismatches to the polynucleotide or a unique segment ofthe polynucleotide of claim
 1. 29. A method of treatment of a subject inneed of enhanced activity or expression of stem cell growth factor-likepolypeptide of claim 10 comprising administering to the subject acomposition selected from the group consisting of: (a) a therapeuticamount of a agonist of said polypeptide; (b) a therapeutic amount of thepolypeptide; and (c) a therapeutic amount of a polynucleotide encodingthe polypeptide in a form and under conditions such that the polypeptideis produced, and a pharmaceutically acceptable carrier.
 30. A method oftreatment of a subject having need to inhibit activity or expression ofstem cell growth factor-like polypeptide of claim 10 comprisingadministering to the subject a composition selected from the groupconsisting of: (a) a therapeutic amount of an antagonist to saidpolypeptide; (b) a therapeutic amount of a polynucleotide that inhibitsthe expression of the nucleotide sequence encoding said polypeptide; and(c) a therapeutic amount of a polypeptide that competes with the stemcell growth factor-like polypeptide for its ligand and apharmaceutically acceptable carrier.